Methods and Compositions for Delivery of Biotin to Mitochondria

ABSTRACT

Certain exemplary embodiments are directed to a biologically active composition of matter (and uses thereof) configured for targeted delivery of biotin to mitochondria, the composition comprising a first D-biotin conjugated to a water-soluble, cell-permeable, peptide sequence, wherein the peptide sequence is selected from a polypeptide group with an alternating aromatic-cationic motif.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submittedelectronically in XML format. The Sequence Listing XML is incorporatedherein by reference. Said XML file, created on Oct. 3, 2022, is named1255-008_SL.xml and is 3,925 bytes in size.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

A wide variety of potential, feasible, and/or useful embodiments will bemore readily understood through the herein-provided, non-limiting,non-exhaustive description of certain exemplary embodiments, withreference to the accompanying exemplary drawings in which:

FIGS. 1A, 1B, and 1C are chemical structure diagrams showing how biotincan be conjugated to mitochondria-targeted peptide sequences.

FIG. 2 are fluorescent microscopic images related to cellular uptake ofcertain exemplary compounds.

FIG. 3 is a graph showing exemplary cellular uptake.

FIG. 4 are fluorescent microscopic images related to cellular uptake ofcertain exemplary compounds.

FIG. 5 is a graph showing exemplary cellular uptake.

FIG. 6 are fluorescent microscopic images related tomitochondria-targeting of certain exemplary compounds.

FIG. 7 is a graph showing exemplary cell viability.

FIG. 8 are microscopic images showing exemplary cell number.

FIG. 9 is a graph showing exemplary cell viability.

FIG. 10 is a graph showing exemplary cellular ATP levels.

FIG. 11 is a graph showing exemplary cellular ATP levels.

FIG. 12 is a graph showing exemplary cellular ATP levels.

FIG. 13 are fluorescent microscopic images showing mitochondrialpotential.

FIG. 14 are fluorescent microscopic images showing exemplarymitochondrial potential.

FIG. 15 are microscopic images of scratch area related to certainexemplary compounds.

FIG. 16 is a graph showing exemplary scratch area.

FIG. 17 is a graph showing exemplary scratch area.

FIG. 18 are microscopic images of scratch area related to certainexemplary compounds.

FIG. 19 is a graph showing exemplary scratch area.

FIG. 20 are microscopic images related to cell proliferation of certainexemplary compounds.

FIG. 21 is a graph showing exemplary cell proliferation.

FIG. 22 are microscopic images related to cell proliferation of certainexemplary compounds.

FIG. 23 is a graph showing exemplary cell proliferation.

FIG. 24 are microscopic images related to mitochondrial potential ofcertain exemplary compounds.

FIG. 25 is a graph showing exemplary mitochondrial potential.

FIG. 26 are fluorescent microscopic images related to retina uptake ofcertain exemplary compound.

FIG. 27 is a fluorescent microscopic image related to retina uptake ofcertain exemplary compound.

DETAILED DESCRIPTION

Certain exemplary embodiments described herein can relate to methods andcompositions for enhancing cellular and/or mitochondrial uptake ofbiotin. Targeted delivery of biotin to mitochondria can improve efficacyand avoid the use of high doses of biotin.

Certain exemplary embodiments described herein can provide a method forusing water-soluble, cell-permeable, mitochondria-targeting peptidesequences to deliver biotin to mitochondria.

Certain exemplary embodiments described herein relate to shortwater-soluble peptide sequences selected from a polypeptide group with ageneral aromatic-cationic motif, meaning that the amino acids of thepolypeptide group can be arranged as, e.g.,[aromatic-cationic-aromatic-cationic] or[cationic-aromatic-cationic-aromatic].

Certain exemplary embodiments described herein can include polypeptidescomposed of naturally occurring amino acids.

Certain exemplary embodiments described herein can include D aminoacids, which can help make the peptide more resistant to hydrolysis bypeptidase enzymes. Certain exemplary embodiments described herein caninclude polypeptides composed of one or more non-naturally occurringamino acids. Non-naturally occurring amino acids are those amino acidsthat typically are not synthesized in normal metabolic processes inliving organisms, and do not naturally occur in proteins. Non-naturallyoccurring amino acids can include derivatives of naturally occurringamino acids.

The peptides useful in the present invention can contain one or morenon-naturally occurring amino acids. The non-naturally occurring aminoacids may be L-, dextrorotatory (D), or mixtures thereof. Optimally, thepeptide has no amino acids that are naturally occurring.

Non-naturally occurring amino acids are those amino acids that do notnaturally occur in proteins. In addition, the non-naturally occurringamino acids useful in the present invention preferably are also notrecognized by common proteases.

The non-naturally occurring amino acid can be present at any position inthe peptide. For example, the non-naturally occurring amino acid can beat the N-terminus, the C-terminus, or at any position between theN-terminus and the C-terminus.

The non-natural amino acids may, for example, comprise alkyl, aryl, oralkylaryl groups. Some examples of alkyl amino acids includeα-aminobutyric acid, β-aminobutyric acid, γ-aminobutyric acid,δ-aminovaleric acid, and ε-aminocaproic acid. Some examples of arylamino acids include ortho-, meta, and para-aminobenzoic acid. Someexamples of alkylaryl amino acids include ortho-, meta-, andpara-aminophenylacetic acid, and γ-phenyl-β-aminobutyric acid.

Non-naturally occurring amino acids also include derivatives ofnaturally occurring amino acids. The derivatives of naturally occurringamino acids may, for example, include the addition of one or morechemical groups to the naturally occurring amino acid.

For example, one or more chemical groups can be added to one or more ofthe 2′, 3′, 4′, 5′, or 6′ position of the aromatic ring of aphenylalanine or tyrosine residue, or the 4′, 5′, 6′, or 7′ position ofthe benzo ring of a tryptophan residue. The group can be any chemicalgroup that can be added to an aromatic ring. Some examples of suchgroups include branched or unbranched C₁-C₄ alkyl, such as methyl,ethyl, n-propyl, isopropyl, butyl, isobutyl, or t-butyl, C₁-C₄ alkyloxy(i.e., alkoxy), amino, C₁-C₄ alkylamino and C₁-C₄ dialkylamino (e.g.,methylamino, dimethylamino), nitro, hydroxyl, halo (i.e., fluoro,chloro, bromo, or iodo). Some specific examples of non-naturallyoccurring derivatives of naturally occurring amino acids includenorvaline (Nva), norleucine (Nle), and hydroxyproline (Hyp).

Another example of a modification of an amino acid in a peptide usefulin the methods of the present invention is the derivatization of acarboxyl group of an aspartic acid or a glutamic acid residue of thepeptide. One example of derivatization is amidation with ammonia or witha primary or secondary amine, e.g. methylamine, ethylamine,dimethylamine or diethylamine. Another example of derivatizationincludes esterification with, for example, methyl or ethyl alcohol.

Another such modification includes derivatization of an amino group of alysine, arginine, or histidine residue. For example, such amino groupscan be acylated. Some suitable acyl groups include, for example, abenzoyl group or an alkanoyl group comprising any of the C₁-C₄ alkylgroups mentioned above, such as an acetyl or propionyl group. Certainexemplary embodiments described herein include amidation of theC-terminus of the peptide sequence, which can help make the peptide moreresistant to hydrolysis by carboxypeptidases.

Carboxyl groups, especially the terminal carboxyl group of a C-terminalamino acid, can be amidated with, for example, ammonia to form theC-terminal amide. Alternatively, the terminal carboxyl group of theC-terminal amino acid may be amidated with any primary or secondaryamine. The primary or secondary amine may, for example, be an alkyl,especially a branched or unbranched C₁-C₄ alkyl, or an aryl amine.Accordingly, the amino acid at the C-terminus of the peptide may beconverted to an amido, N-methylamido, N-ethylamido, N,N-dimethylamido,N,N-diethylamido, N-methyl-N-ethylamido, N-phenylamido orN-phenyl-N-ethylamido group.

The free carboxylate groups of the asparagine, glutamine, aspartic acid,and glutamic acid residues not occurring at the C-terminus of thearomatic-cationic peptides of the present invention may also be amidatedwherever they occur within the peptide. The amidation at these internalpositions may be with ammonia or any of the primary or secondary aminesdescribed above.

Certain exemplary embodiments described herein can include polypeptidescomposed of a minimum of four amino acids.

Certain exemplary embodiments described herein can include polypeptidescomposed of no more than six amino acids.

Certain exemplary embodiments described herein are directed towater-soluble peptide sequences selected from polypeptides composed of4-6 amino acids (in either D or L configuration) with an alternatingaromatic-cationic motif.

Certain exemplary embodiments described herein are directed towater-soluble peptide sequences including, but not limited to:

(SS-31) D-Arg-L-(2′6′-dimethylTyr)-L-Lys-L-Phe-NH₂ (SPN02)D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys-NH₂; (SPN07) D-Trp-D-Arg-D-Trp-D-Lys-OH;(SPN10) (SEQ ID NO: 1) L-Trp-L-Arg-L-Trp-L-Lys-NH₂; (SPN13)D-Trp-D-Arg-D-Trp-D-Lys-NH₂; and (SPN14) (SEQ ID NO: 2)L-Trp-L-Arg-L-Trp-L-Lys-OH.

Certain exemplary embodiments described herein can provide methods forconjugating D-biotin to short (i.e., 4 to 6 amino acids), water-soluble,cell-permeable, mitochondria-targeting peptide sequences for targeteddelivery of biotin to mitochondria.

Certain exemplary peptide sequences described herein are defined by anN-terminus and a C-terminus, and can comprise an α-amine at theN-terminus. Certain exemplary embodiments described herein can providemethods for conjugating D-biotin to the N-terminus α-amine ofmitochondria-targeting peptide sequences.

Certain exemplary peptide sequences described herein can comprise alysine residue, having an ε-amine, at their C-terminus. Certainexemplary embodiments described herein can provide methods forconjugating D-biotin to the ε-amine of lysine residues(ε-N-[d-biotinyl]-L-lysine) at the C-terminus of themitochondria-targeting peptide sequences.

Certain exemplary embodiments described herein can provide methods forconjugating D-biotin to both the N-terminus α-amine and to the ε-amineon lysine residues at the C-terminus of the mitochondria-targetingpeptide sequences.

Certain exemplary embodiments described herein are directed to at leastone therapeutically effective composition of matter and/or methods formaking and/or using such a composition and/or one or more of itscomponents, the composition comprising:

one or more compounds selected from a polypeptide group consisting of:

(SPN05) D-Biotin-D-Arg-L-(2′6′-dimethylTyr)-L-Lys-L- Phe-NH₂; (SPN08)D-Biotin-D-Trp-D-Arg-D-Trp-D-Lys-OH; (SPN09)D-Trp-D-Arg-D-Trp-D-Lys(biotinyl)-OH; (SPN11)L-Trp-L-Arg-L-Trp-L-Lys(biotinyl)-NH₂; (SPN12)D-Biotin-L-Trp-L-Arg-L-Trp-L-Lys(biotinyl)-NH₂; (SPN15)D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys(biotinyl)-NH₂; and (SPN16)D-Biotin-D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys(biotinyl)- NH₂.

One or more exemplary compositions described herein can comprise one ormore peptide-conjugated biotin molecules adapted to stimulate cellularATP production. An exemplary composition can include vitamins and/oramino acids and/or metabolic supplements in effective concentrationsthat promote ATP synthesis.

One or more exemplary compositions described herein can comprise one ormore peptide-conjugated biotin molecules adapted to preservemitochondrial potential.

Certain exemplary embodiments described herein relate to novelcompositions and/or methods adapted for promoting cell survival whencells are cultured ex vivo in the absence of serum. One or moreexemplary compositions described herein can comprise one or morepeptide-conjugated biotin molecules adapted to stimulate mitochondrialfunction, such as to promote cell proliferation. One or more exemplarycompositions described herein can include vitamins and/or amino acidsand/or metabolic supplements in effective concentrations that exhibitadditive or synergistic activity in maintaining cell growth.

To perform cell therapy, because of the small number of stem cells,extensive ex vivo expansion is required to obtain sufficient cellnumbers for treatment of a clinical indication. Media containing fetalbovine serum (FBS) provides a supportive environment for isolation andexpansion of mesenchymal stem cells. FBS provides attachment factors,growth factors and a host of other nutrients. Yet, in addition to theinherent variability in FBS and a limited global supply, serum productsalso can be a source of pathogens and/or contain serum proteins thathave the potential to elicit immune response in human recipients.

Serum depletion causes cell cycle arrest and apoptosis which severelylimits the yield of stem cells or primary cells for clinical use. Avariety of growth factors must be added to serum-free media to permitstem cell expansion, and this often involves the use of human-sourcedsupplements that might be contaminated with human pathogens. Certainexemplary media described herein comprise chemically defined yetserum-free and xeno-free constituents that support the growth andattachment of human primary cells and passaged cultures to allow largescale production of primary mammalian cells for clinical use.

Large-scale production of primary mammalian cells can be crucial forlaboratory production of meat as an alternative to traditionallivestock-derived meats. The culturing of animal myoblasts with FBS isnot sustainable. Certain exemplary media described herein comprisechemically defined, serum-free constituents that support theproliferation of animal myoblasts for large scale production oflab-grown meats.

Certain exemplary embodiments described herein relate to novelcompositions and/or methods adapted for promoting cell or tissuesurvival for transplantation in humans. One or more exemplarycompositions described herein can comprise one or morepeptide-conjugated biotin molecules adapted to stimulate mitochondrialcellular ATP production to promote cell survival. One or more exemplarycompositions described herein can include vitamins and/or amino acidsand/or metabolic supplements in effective concentrations that exhibitadditive or synergistic activity in maintaining organ survival.

Pancreatic islet transplantation is an approach to b-cell replacementtherapy for type 1 diabetics. The addition of one or morepeptide-conjugated biotin molecules that can improve cell viabilitythroughout the harvesting and purification procedure, can greatlyimprove islet yield, and/or increase the success of glycemic control inthe recipient.

Ischemia and hypoxia are inevitable events during preservation of organsprior to transplantation. Once the organ has been deprived of normalblood supply, depletion of mitochondrial ATP synthesis leads to celldeath. The duration of storage varies from 4-6 hours for heart and lungsand up to 36 hours for kidneys. The addition of one or morepeptide-conjugated biotin molecules that can improve cell viability inorgan preservation solutions can prolong survival time of an organ topermit broader distribution to matching recipients. Improved quality oftransplant organs can also reduce delayed graft function and graftfailure.

Certain exemplary embodiments described herein relate to novelcompositions and/or methods adapted for promoting tissue health and/orpreventing tissue injuries. Certain exemplary methods described hereinprovide for systemic administration of an exemplary composition to themammal. The composition can comprise one or more peptide-conjugatedbiotin molecules adapted to stimulate mitochondrial cellular ATPproduction to promote cellular function. The composition can includevitamins and/or amino acids and/or metabolic supplements in effectiveconcentrations that exhibit additive or synergistic activity inmaintaining tissue health.

Aging is associated with decreased proliferative ability of many celltypes, including skin and epithelial membranes. The intestinalepithelium completely self-renews within 5 days, while the lungepithelium can take as long as 6 months to renew. Aging diminishes thecapacity of epithelial regeneration and leads to progressive epithelialinjury.

Most tissues exhibit a progressive decline in regeneration capabilitywith age that results in tissue degeneration, malfunction, andpathology. Certain methods described herein can promote tissue healthduring aging can prevent and/or reduce many of these age-relatedfunctional disabilities.

Exemplary methods described herein relate to novel compositions and/ormethods adapted for promoting tissue health that can promoteproliferation of resident stem/progenitor cells in vivo in aged and/ordamaged tissues.

Certain exemplary embodiments described herein relate to novelcompositions and/or methods adapted for promoting tissue repair andregeneration. Certain exemplary methods described herein provide forsystemic administration of an exemplary composition to the mammal aftertissue injury. The composition can comprise one or morepeptide-conjugated biotin molecules adapted to stimulate mitochondrialcellular ATP production to promote cell proliferation and/or tissueregeneration. The composition can include vitamins and/or amino acidsand/or metabolic supplements in effective concentrations that exhibitadditive or synergistic activity in maintaining tissue health.

Tissue injury can include injury to skin, and/or soft tissues (e.g.,muscles, tendons, ligaments, nerves, blood vessels), and/or hard tissues(e.g., bones, teeth), and/or solid organs (e.g., heart, kidneys, lungs,liver, spleen, intestines, etc.). The cause of tissue injury caninclude, e.g., trauma, hypoxia (i.e., low oxygen supply), ischemia(i.e., low blood flow), infectious agents, drugs, chemicals, and/ortoxins, etc.

Certain exemplary embodiments described herein relate to novelcompositions and/or methods adapted for the treatment of wounds and/ortissue injury, and/or for the promotion of tissue regeneration and morerapid wound repair. Certain exemplary methods described herein providefor application of a composition directly to a wound, such as tostimulate (i.e., promote) adjacent cells in the periphery of the woundto proliferate (i.e., increase in number of cells), to facilitate and/orachieve wound closure. The composition can comprise one or morepeptide-conjugated biotin molecules adapted to stimulate mitochondrialcellular ATP production to promote cell proliferation and/or migration(i.e., cell movement). The composition can include vitamins and/orsupplements and/or metabolic supplements in effective concentrationsthat exhibit additive or synergistic activity in stimulating woundhealing, such as for post-surgical wounds and/or non-healing chronicwounds, e.g., pressure ulcers and/or diabetic ulcers and/or venousulcers in patients.

Exemplary methods described herein relate to novel compositions and/ormethods adapted for the treatment of tissue injury that can promoteproliferation of resident stem/progenitor cells in vivo in damagedtissues.

Certain exemplary embodiments can provide a pharmaceutical compositionthat can be useful in the promotion of tissue repair and/orregeneration, that composition comprising:

-   -   (a) one or more compounds that promote ATP production and cell        proliferation;    -   (b) at least one vitamin; and/or    -   (c) at least one amino acid; and/or    -   (d) at least one metabolic supplement

Certain exemplary embodiments described herein can provide a method ofenhancing mitochondrial ATP production in a mammal, the methodcomprising administering to the mammal a therapeutically effectiveamount of a compound having the formula:

(SPN05) D-Biotin-D-Arg-L-(2′6′-dimethylTyr)-L-Lys-L- Phe-NH₂; (SPN08)D-Biotin-D-Trp-D-Arg-D-Trp-D-Lys-OH; (SPN09)D-Trp-D-Arg-D-Trp-D-Lys(biotinyl)-OH; (SPN11)L-Trp-L-Arg-L-Trp-L-Lys(biotinyl)-NH₂; (SPN12)D-Biotin-L-Trp-L-Arg-L-Trp-L-Lys(biotinyl)-NH₂; (SPN15)D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys(biotinyl)-NH₂; and/or (SPN16)D-Biotin-D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys(biotinyl)- NH₂

wherein the compound is administered to the mammal as a compositioncomprising a pharmaceutically acceptable carrier.

Certain exemplary embodiments described herein can provide a compositionconfigured for increasing cell proliferation for primary cells culturedex vivo without serum, the composition comprising of an activeingredient that comprises one or more peptide-conjugated biotinmolecules that stimulate mitochondrial ATP production and cellproliferation, and a second ingredient that compromises at least onevitamin and/or amino acid and/or metabolic supplement that potentiatesATP production and cell proliferation.

Certain exemplary embodiments described herein can provide a compositionconfigured for increasing cell and organ survival ex vivo, thecomposition comprising of an active ingredient that comprises one ormore peptide-conjugated biotin molecules that stimulate mitochondrialATP production and cell proliferation, and a second ingredient thatcompromises at least one vitamin and/or amino acid and/or metabolicsupplement that potentiates ATP production and cell proliferation.

Certain exemplary embodiments described herein can provide a compositionconfigured for increasing cell proliferation, such as in a wound area,the composition comprising an active ingredient that comprises one ormore peptide-conjugated biotin molecules and/or a second ingredient thatcompromises an admixture of at least one vitamin and/or one amino acidand/or one metabolic supplement that potentiates cell proliferation andmigration.

Examples of amino acids that can be used with certain exemplaryembodiments described herein include L-isomers of all natural aminoacids including essential and non-essential amino acids (isoleucine,leucine, alanine, asparagine, lysine, aspartic acid, methionine,cysteine, phenylalanine, glutamic acid, threonine, glutamine,tryptophan, glycine, valine, proline, serine, tyrosine, arginine,histidine), as well as taurine that is naturally derived from cysteine.

Examples of metabolic supplements that can be used with certainexemplary embodiments described herein include pyruvate, carnitine,acetylcarnitine, creatine, α-ketoglutarate, α-lipoic acid, coenzyme Q₁₀,nicotinamide riboside, nicotinamide mononucleotide.

Certain exemplary embodiments described herein can provide a method ofenhancing survival and proliferation of primary mammalian cells inserum-free, chemically-defined media by adding a formulation comprisingan effective amount of one of more peptide-conjugated biotin moleculesand a mixture of at least one vitamin and/or one amino acid and/or onemetabolic supplement to the culture media.

In certain exemplary embodiments of one or more methods describedherein, the primary cells can include bone marrow stem cells ormesenchymal stem cells for autologous, allogeneic, or xenogenicregenerative medicine applications.

In certain exemplary embodiments of one or more methods describedherein, the primary cells can be stem cells obtained from placenta orumbilical cord blood for allogeneic transplant.

In certain exemplary embodiments of one or more methods describedherein, the primary cells can be hematopoietic cells such as T cells forchimeric antigen receptor (CAR) T-cell therapy.

In certain exemplary embodiments of one or more methods describedherein, the primary cells can be mammalian cells cultured for theproduction of therapeutic proteins such as monoclonal antibodies and/orbiopharmaceuticals and/or in the development and/or production of viralvaccines.

In certain exemplary embodiments of one more methods described herein,the primary cells can be animal cells such as myoblasts cultured for invitro production of laboratory-grown, slaughter-free meat.

In certain exemplary embodiments of one or more methods describedherein, the primary cells can be pancreatic islet cells harvested from adonor pancreas and purified in a laboratory before transplantation.

Certain exemplary embodiments described herein can provide a method foroptimizing and/or improving stem cell transplantation and/ormitochondrial transfer for tissue regeneration by treating the stemcells and/or mitochondria with a solution comprising a therapeuticallyeffective amount of one or more peptide-conjugated biotin molecules anda mixture of at least one vitamin and/or one amino acid and/or onemetabolic supplement prior to transplantation.

In certain exemplary embodiments of one or more methods describedherein, the cells can be resident stem/progenitor cells in injuredtissues and one or more peptide-conjugated biotin molecules and amixture of at least one vitamin and/or one amino acid and/or onemetabolic supplement can be administered systemically to the subject.

In certain exemplary embodiments of one or more methods describedherein, one or more peptide-conjugated biotin molecules and at least onevitamin and/or one amino acid and/or one metabolic supplement can beadministered systemically to the subject after mitochondrialtransplantation to optimize survival and function of the mitochondria.

In certain exemplary embodiments of one or more methods describedherein, one or more peptide-conjugated biotin molecules and at least onevitamin and/or one amino acid and/or one metabolic supplement can beadded to serum-free medium for cultivation of lab-grown meat to replacelivestock-derived meat.

Certain exemplary embodiments described herein can provide a method ofenhancing organ preservation solutions by adding to the preservationsolution a formulation comprising an effective amount of one or morepeptide-conjugated biotin molecules and at least one vitamin and/or oneamino acid and/or one metabolic supplement.

In certain exemplary embodiments of one or more methods describedherein, the organ can include kidney, liver, heart, lungs, pancreas,skin, intestines, cornea, trachea, and/or blood vessels.

In certain exemplary embodiments of one or more methods describedherein, an effective amount of one or more peptide-conjugated biotinmolecules and at least one vitamin and/or one amino acid and/or onemetabolic supplement can be administered systemically to the transplantrecipient to improve early graft function and improve graft survival.

Certain exemplary embodiments described herein can provide a method ofenhancing tissue health in a subject by administering a micronutrientformulation comprising an effective amount of one or morepeptide-conjugated biotin molecules and a mixture of at least onevitamin and/or one amino acid and/or one metabolic supplement.

Certain exemplary embodiments described herein can provide a method ofenhancing tissue health in a subject by administering a micronutrientformulation comprising an effective amount of one or morepeptide-conjugated biotin molecules and a mixture of acetylcarnitineand/or α-ketoglutarate.

Certain exemplary embodiments described herein can provide a method ofenhancing tissue health in a subject by giving a micronutrientformulation comprising an effective amount of one or morepeptide-conjugated biotin molecules and a mixture of acetylcarnitine,α-ketoglutarate, and/or taurine.

In certain exemplary embodiments of one or more methods describedherein, tissue health can include health of keratinous tissues (e.g.skin, hair, and/or nails), and/or muscle, and/or joints, and/or bone,and/or heart, and/or lung, and/or kidney, and/or brain, and/or vision,and/or hearing.

In certain exemplary embodiments of one or more methods describedherein, the tissue injury can be caused by aging. All cells canexperience changes with aging. Many cells can lose their ability tofunction, waste products can accumulate in cells, connective tissues canbecome stiff, and many tissues can lose mass.

Certain exemplary embodiments described herein can provide a method ofreducing, mitigating, and/or reversing age-related injury in a subjectby administering a composition comprising an effective amount of one ormore peptide-conjugated biotin molecules and at least one vitamin and/orone amino acid and/or one metabolic supplement, that administrationoccurring orally, sublingually, and/or subcutaneously, etc. Apharmaceutical preparation for oral administration can be a solution,suspension, or solid forms, such as tablets, capsules, and powders, etc.A pharmaceutical preparation for sublingual administration or forsubcutaneous injection can be prepared by mixing such a composition withnon-toxic, therapeutically-inert, and/or liquid carriers customarilyused in sublingual and/or subcutaneous preparations.

Certain exemplary embodiments described herein can provide a method ofenhancing skin wound healing in a subject by administering a topicalsolution comprising a therapeutically effective amount of one or morepeptide-conjugated biotin molecules and at least one vitamin and/or oneamino acid and/or one metabolic supplement directly to the wound area.

Certain exemplary embodiments described herein can provide a method ofenhancing skin wound healing in a subject by giving a topical solutioncompromising a therapeutically effective amount of one of morepeptide-conjugated biotin molecules and taurine directly to the woundarea.

In certain exemplary embodiments of one or more methods describedherein, the wound can be pressure wound, surgical wound, burn wound,trauma, and/or wounds that have been exposed to one or more chemicalsand/or therapeutic radiation, etc.

For topical administration to the skin, certain exemplary compositionsdescribed herein can be prepared as a spray, ointment, cream, and/orgel. A pharmaceutical preparation for topical administration to the skincan be prepared by mixing such a composition with non-toxic,therapeutically-inert, solid, and/or liquid carriers customarily used intopically-administered pharmaceutical preparations.

Certain exemplary embodiments described herein can provide a method ofenhancing gingival and/or periodontal healing in a subject byadministering a topical solution comprising a therapeutically effectiveamount of one or more peptide-conjugated biotin molecules and at leastone vitamin and/or one amino acid and/or one metabolic supplementdirectly to the wound area.

Certain exemplary embodiments described herein can provide a method ofenhancing gingival and/or periodontal healing in a subject byadministering a topical solution comprising a therapeutically effectiveamount of one or more peptide-conjugated biotin molecules and taurine.

For topical administration to the oral mucosal membrane, certainexemplary compositions described herein can be prepared as a spray,ointment, gel, mouth wash, and/or toothpaste, etc. A pharmaceuticalpreparation for topical administration to the mucosal membrane can beprepared by mixing such a composition with non-toxic,therapeutically-inert, and/or liquid carriers customarily used intopically-administered pharmaceutical preparations.

Certain exemplary embodiments described herein can provide a method ofenhancing repair to injuries of the eye in a subject by administering,directly to the eye, a topical solution comprising a therapeuticallyeffective amount of one or more peptide-conjugated biotin molecules andat least one vitamin and/or one amino acid and/or one metabolicsupplement.

Certain exemplary embodiments described herein can provide a method ofenhancing repair to injuries of the eye in a subject by administering atopical solution comprising a therapeutically effective amount of one ormore peptide-conjugated biotin molecules and taurine.

In certain exemplary embodiments of one or more methods describedherein, the injury to the eye can be one or more of, e.g., acute cornealabrasion, subconjunctival hemorrhages, and/or retinal detachment, etc.,and/or chronic eye diseases including, e.g., age-related maculardegeneration, diabetic retinopathy, glaucoma, and/or dry eye disease,etc.

Certain exemplary embodiments can provide a method of enhancing bone andsoft tissue healing in a subject by administering a solution comprisinga therapeutically effective amount of one or more peptide-conjugatedbiotin molecules and at least one vitamin and/or one amino acid and/orone metabolic supplement directly to the wound area.

In certain exemplary embodiments of one or more methods describedherein, the injury can be acute and/or chronic, such as trauma,arthritis, tendinitis, one or more ligament tears, and/or nervecompression, etc.

For direct application to soft tissues, certain exemplary compositionsdescribed herein can be prepared as a sterile solution for injectioninto one or more joints, tendons, muscles, and/or nerves, etc. Apharmaceutical preparation for injection can be prepared by mixing sucha composition with non-toxic, therapeutically-inert, and/or liquidcarriers customarily used in such preparations, such aspolyethyleneglycol and hyaluronic acid.

Certain exemplary embodiments described herein can provide a method ofenhancing organ repair in a subject by administering a solutioncomprising a therapeutically effective amount of one or morepeptide-conjugated biotin molecules and at least one vitamin and/or oneamino acid and/or one metabolic supplement, such as intravenously,intramuscularly, subcutaneously, and/or orally, etc. A pharmaceuticalpreparation for injection can be prepared by mixing such a compositionwith non-toxic, therapeutically-inert, and/or liquid carrierscustomarily used in injectable pharmaceutical preparations. Apharmaceutical preparation for oral administration can be administeredas, e.g., a solution, suspension, and/or solid form, such as one or moretablets, capsules, and/or powders, etc.

In certain exemplary embodiments of one or more methods describedherein, the tissue injury can be caused by acute diseases, e.g., trauma,reduced blood flow, reduced oxygen supply, infectious agents, drugs,and/or toxins to one or more organs, structures, and/or systems, such asthe heart, brain, kidney, liver, intestines, and/or limbs, etc.

In certain exemplary embodiments of one or more methods describedherein, the tissue injury can be caused by chronic diseases, e.g., heartfailure, chronic kidney disease, inflammatory bowel disease, diabeticcomplications, stroke, macular degeneration, and/or neurodegenerativediseases including Parkinson's Disease, Amyotropic Lateral Sclerosis,Huntington's Disease, Chronic Traumatic Encephalopathy, and/orAlzheimer's Disease, etc.

In certain exemplary embodiments of one or more methods describedherein, a mixture of one or more peptide-conjugated biotin molecules canbe administered to a subject with progressive multiple sclerosis,frontotemporal dementia, Parkinson's disease, and/or Alzheimer'sdisease.

In certain exemplary embodiments of one or more methods describedherein, a mixture of one or more peptide-conjugated biotin molecules andthiamine can be administered to a subject withbiotin-thiamine-responsive basal ganglia disease.

In certain exemplary embodiments of one or more methods describedherein, a mixture of one or more peptide-conjugated biotin molecules canbe administered to a subject with inflammatory bowel disease.

Example 1—Short Aromatic-Cationic Peptide Sequences Increase CellularUptake of Biotin

Certain peptide sequences described herein can be water-solublepolypeptides composed of 4 to 6 amino acids that can have a generalaromatic-cationic motif, meaning that the peptide sequence can be, e.g.,[aromatic-cationic-aromatic-cationic] or[cationic-aromatic-cationic-aromatic].

Certain “short” peptide sequences described herein can be polypeptidescomposed of a minimum of four amino acids and a maximum of six aminoacids.

The amino acids can be naturally occurring. Naturally occurring aminoacids include the twenty most common amino acids normally found inproteins, i.e., alanine (Ala), arginine (Arg), asparagine (Asn),aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid(Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu),lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro),serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), andvaline (Val). Aromatic amino acids can include Phe, Tyr, and Trp.Cationic amino acids can include Lys, Arg, and His. The amino acids caninclude the natural amino acids in the D-configuration. In certainpeptides, the carboxyl terminus can be amidated.

The amino acids can be non-naturally occurring. Non-naturally occurringamino acids are those amino acids that typically are not synthesized innormal metabolic processes in living organisms, and do not naturallyoccur in proteins. Non-naturally occurring amino acids can includederivatives of naturally occurring amino acids, in either L- orD-configuration.

Certain exemplary embodiments can provide for administering to thesubject a composition for therapeutic purposes. In certain exemplarytherapeutic applications, compositions and/or medicaments can beadministered to a subject suspected of, or already suffering from, sucha disease and/or condition in an amount sufficient to cure, or at leastpartially arrest, the symptoms of the disease and/or condition,including its complications and intermediate pathological phenotypes indevelopment of the disease and/or condition.

In certain exemplary embodiments, therapeutic methods compriseadministration of the composition in conjunction with one or more activeagents. In certain exemplary embodiments, peptide administration ischronic.

In certain exemplary embodiments the peptide can be administered inconjunction with one or more thrombolytic agents. In certain exemplaryembodiments, the one or more thrombolytic agents can be selected fromthe group consisting of: tissue plasminogen activator, urokinase,prourokinase, streptokinase, acylated form of plasminogen, acylated formof plasmin, and acylated streptokinase-plasminogen complex.

In certain exemplary embodiments, therapeutic methods can compriseadministration of the composition in conjunction with one or moreantihypertensive agents. In certain exemplary embodiments, the one ormore antihypertensive agents can comprise diuretics, adrenergic receptorantagonists, calcium channel blockers, renin inhibitors, angiotensinconverting enzyme (ACE) inhibitors, angiotensin II receptor antagonists,aldosterone antagonists, vasodilators, and/or alpha-2 agonists.

In certain exemplary embodiments, the diuretics can comprise loopdiuretics, thiazide diuretics, thiazide-like diuretics, and/orpotassium-sparing diuretics. In certain exemplary embodiments, thediuretics can comprise bumetanide, ethacrynic acid, furosemide,torsemide, epitizide, hydrochlorothiazide, chlorothiazide,bendroflumethiazide, indapamide, chlorthalidon, metolazone, amiloride,triamterene, and/or spironolactone.

In certain exemplary embodiments, the adrenergic receptor antagonistscan comprise beta blockers, alpha blockers, or mixed alpha and betablockers. In certain exemplary embodiments, the adrenergic receptorantagonists can comprise atenolol, metoprolol, nadolol, oxprenolol,pindolol, propranolol, timolol, doxazosin, phentolamine, indoramin,phenoxybenzamine, prazosin, terazosin, tolazoline, bucindolol,carvedilol, and/or labetalol.

In certain exemplary embodiments, the calcium channel blockers cancomprise dihydropyridines and/or non-dihydropyridines. In certainexemplary embodiments, the calcium channel blockers can compriseamlodipine, felodipine, isradipine, lercanidipine, nicardipine,nifedipine, nimodipine, nitrendipine, diltiazem, and/or verapamil.

In certain exemplary embodiments, the renin inhibitors can compriseAliskiren®.

In certain exemplary embodiments, the angiotensin converting enzyme(ACE) inhibitors can comprise captopril, enalapril, fosinopril,lisinopril, perindopril, quinapril, ramipril, trandolapril, and/orbenazepril.

In certain exemplary embodiments, the angiotensin II receptorantagonists can comprise Irbesartan®.

In certain exemplary embodiments, the aldosterone antagonists cancomprise eplerenone and/or spironolactone.

In certain exemplary embodiments, the vasodilators antagonists cancomprise sodium nitroprusside and/or hydralazine.

In certain exemplary embodiments, the alpha-2 agonists antagonists cancomprise clonidine, guanabenz, methyldopa, moxonidine, guanethidine,and/or reserpine.

In certain exemplary embodiments, any method known to those in the artfor contacting a cell, organ, and/or tissue with a peptide can beemployed. Suitable methods can include in vitro, ex vivo, and/or in vivomethods. When used in vivo for therapy, the compositions can beadministered to the subject in effective amounts (i.e., amounts thathave desired therapeutic effect). The dose and dosage regimen can dependupon the degree of the injury in the subject, the characteristics of theparticular composition used, e.g., its therapeutic index, the subject,and/or the subject's history.

The effective amount can be determined during pre-clinical trials and/orclinical trials by methods familiar to physicians and/or clinicians. Aneffective amount of a peptide useful in the methods can be administeredto a mammal in need thereof by any of a number of well-known methods foradministering pharmaceutical compounds. The peptide can be administeredsystemically and/or locally.

The peptide may be formulated as a pharmaceutically acceptable salt.Pharmaceutically acceptable salts can be derived from pharmaceuticallyacceptable inorganic or organic bases and from pharmaceuticallyacceptable inorganic or organic acids. When a peptide contains both abasic moiety, such as an amine, pyridine or imidazole, and an acidicmoiety such as a carboxylic acid or tetrazole, zwitterions can be formedand are included within the term “salt” as used herein. Salts derivedfrom pharmaceutically acceptable inorganic bases include ammonium,calcium, copper, ferric, ferrous, lithium, magnesium, manganic,manganous, potassium, sodium, and zinc salts, and the like. Saltsderived from pharmaceutically acceptable organic bases include salts ofprimary, secondary and tertiary amines, including substituted amines,cyclic amines, naturally-occurring amines and the like, such asarginine, betaine, caffeine, choline, N,N′-dibenzylethylenediamine,diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol,ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine,glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperadine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine, and the like. Salts derived frompharmaceutically acceptable inorganic acids include salts of boric,carbonic, hydrohalic (hydrobromic, hydrochloric, hydrofluoric orhydroiodic), nitric, phosphoric, sulfamic, and sulfuric acids. Saltsderived from pharmaceutically acceptable organic acids include salts ofaliphatic hydroxyl acids (e.g., citric, gluconic, glycolic, lactic,lactobionic, malic, and tartaric acids), aliphatic monocarboxylic acids(e.g., acetic, butyric, formic, propionic and trifluoroacetic acids),amino acids (e.g., aspartic and glutamic acids), aromatic carboxylicacids (e.g., benzoic, p-chlorobenzoic, diphenylacetic, gentisic,hippuric, and triphenylacetic acids), aromatic hydroxyl acids (e.g.,o-hydroxybenzoic, p-hydroxybenzoic, 1-hydroxynaphthalene-2-carboxylicand 3-hydroxynaphthalene carboxylic acids), ascorbic, dicarboxylic acids(e.g., fumaric, maleic, oxalic and succinic acids), glucoronic,mandelic, mucic, nicotinic, orotic, pamoic, pantothenic, sulfonic acids(e.g., benzenesulfonic, camphosulfonic, edisylic, ethanesulfonic,isethionic, methanesulfonic, naphthalenesulfonic,naphthalene-1,5-disulfonic, naphthalene-2,6-disulfonic andp-toluenesulfonic acids), xinafoic acid, and the like. In someembodiments, the salt is an acetate salt or a trifluoroacetate salt.

The compositions described herein, or pharmaceutically acceptable saltsthereof, such as acetate salt or trifluoroacetate salt, can beincorporated into pharmaceutical compositions for administration, singlyor in combination, to a subject for the treatment and/or prevention of adisorder described herein. Such compositions can include the activeagent and a pharmaceutically acceptable carrier, which can include oneor more of saline, solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Supplementaryactive compounds also can be incorporated into certain exemplarycompositions.

Pharmaceutical compositions can be formulated to be compatible with itsintended route of administration. Exemplary routes of administrationinclude parenteral (e.g., intravenous, intradermal, intraperitoneal orsubcutaneous), oral, sublingual, nasal, inhalation, transdermal(topical), intraocular, iontophoretic, and transmucosal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include any one or more of the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates, or phosphates; and agents for theadjustment of tonicity such as sodium chloride or dextrose. In certainexemplary embodiments, pH can be adjusted with acids or bases, such ashydrochloric acid or sodium hydroxide. The parenteral preparation can beenclosed in ampoules, disposable syringes, and/or multiple dose vialsmade of glass and/or plastic. For convenience of the patient and/ortreating physician, the dosing formulation can be provided in a kitcontaining any or all necessary equipment (e.g., vials of drug, vials ofdiluent, syringes, and/or needles, etc.) for a treatment course (e.g., 7days of treatment).

Pharmaceutical compositions suitable for injectable use can includesterile aqueous solutions (where water soluble) and/or dispersionsand/or sterile powders for the extemporaneous preparation of sterileinjectable solutions and/or dispersion. For intravenous administration,suitable carriers can include physiological saline, bacteriostaticwater, Cremophor EL™ (BASF, Parsippany, N.J.), and/or phosphate bufferedsaline (PBS). A composition for parenteral administration can be sterileand/or can be fluid for easy syringability. Certain exemplarycompositions can be stable under the conditions of manufacture and/orstorage and/or can be preserved against the contaminating action ofmicroorganisms such as bacteria and/or fungi.

The composition compositions can include a carrier, which can be asolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and/or liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion, and/or by the use of surfactants. Prevention ofthe action of microorganisms can be achieved by various antibacterialand/or antifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, and the like. Glutathione and/or other antioxidants canbe included to prevent oxidation. Certain exemplary compositions caninclude isotonic agents, for example, sugars and/or polyalcohols such asmannitol, sorbitol, and/or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent that delays absorption, such as aluminummonostearate and/or gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Dispersions can be prepared by incorporating theactive compound into a sterile vehicle that contains a basic dispersionmedium and other desired ingredients, such as one or more of thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, exemplary methods of preparation includevacuum drying and/or freeze drying, which can yield a powder of theactive ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Oral compositions can include an inert diluent and/or an edible carrier.For the purpose of oral therapeutic administration, the active compoundcan be incorporated with excipients and/or used in the form of tablets,troches, or capsules, e.g., gelatin capsules and/or powder dissolvablein a diluent such as water. Oral compositions can be prepared using afluid carrier for use as a mouthwash. Pharmaceutically compatiblebinding agents and/or adjuvant materials can be included as part of thecomposition. Exemplary tablets, pills, capsules, troches and the likecan contain any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth,and/or gelatin; an excipient such as starch and/or lactose, adisintegrating agent such as alginic acid, Primogel, and/or corn starch;a lubricant such as magnesium stearate and/or Sterotes; a glidant suchas colloidal silicon dioxide; a sweetening agent such as sucrose and/orsaccharin; or a flavoring agent such as peppermint, methyl salicylate,and/or orange flavoring; etc.

For administration by inhalation, the compounds can be delivered in theform of an aerosol spray from a pressurized container and/or dispenserthat contains a suitable propellant, e.g., a gas such as carbon dioxide,and/or a nebulizer.

Systemic administration of a therapeutic compound as described hereincan be by transmucosal and/or transdermal means. For transmucosal ortransdermal administration, penetrants appropriate to the barrier to bepermeated can used in the formulation. Such penetrants can include, forexample, for transmucosal administration, detergents, bile salts, and/orfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays. For transdermaladministration, the active compounds can be formulated into ointments,salves, gels, and/or creams. In certain exemplary embodiments,transdermal administration can be performed by iontophoresis,microneedles, and/or electroporation.

A therapeutic protein and/or peptide can be formulated in a carriersystem. The carrier can be a colloidal system. The colloidal system canbe a liposome and/or a phospholipid bilayer vehicle. In certainexemplary embodiments, the therapeutic peptide can be encapsulated in aliposome while maintaining peptide integrity. An active agent can beloaded into a particle prepared from pharmaceutically acceptableingredients including, but not limited to, soluble, insoluble,permeable, impermeable, biodegradable, and/or gastroretentive polymersand/or liposomes. Such particles can include nanoparticles,biodegradable nanoparticles, microparticles, biodegradablemicroparticles, nanospheres, biodegradable nanospheres, microspheres,biodegradable microspheres, capsules, emulsions, liposomes, micelles,and/or viral vector systems.

The carrier can be a polymer, e.g., a biodegradable and/or biocompatiblepolymer matrix. In certain exemplary embodiments, the therapeuticpeptide can be embedded in the polymer matrix, while maintaining proteinintegrity. The polymer can be natural, such as polypeptides, proteins,or polysaccharides, or synthetic, such as poly α-hydroxy acids. Examplesinclude carriers made of, e.g., collagen, fibronectin, elastin,cellulose acetate, cellulose nitrate, polysaccharide, fibrin, gelatin,and combinations thereof. In certain exemplary embodiments, the polymercan be poly-lactic acid (PLA) and/or copoly lactic/glycolic acid (PGLA).The polymeric matrices can be prepared and/or isolated in a variety offorms and/or sizes, including microspheres and nanospheres. Polymerformulations can lead to prolonged duration of therapeutic effect.

In certain exemplary embodiments, the therapeutic compounds can beprepared with carriers that will protect the therapeutic compoundsagainst rapid elimination from the body, such as a controlled releaseformulation, including implants and microencapsulated delivery systems.Biodegradable and/or biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and/or polylacetic acid, etc. Such formulations can beprepared using known techniques. The materials can also be obtainedcommercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc.Liposomal suspensions (including liposomes targeted to specific cellswith monoclonal antibodies to cell-specific antigens) can be used aspharmaceutically acceptable carriers.

The therapeutic compounds can be formulated to enhance intracellulardelivery. For example, liposomal delivery systems and/or fusogenicliposomes can be used to deliver a protein to cells in vivo and/or invitro.

Dosage, toxicity, and therapeutic efficacy of the therapeutic agents canbe determined by standard pharmaceutical procedures in cell culturesand/or experimental animals, e.g., for determining the LD50 (the doselethal to 50% of the population) and/or the ED50 (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects, which is called the “therapeuticindex”, can be expressed as the ratio LD50/ED50. Compounds that exhibithigh therapeutic indices can be preferred. While compounds that exhibittoxic side effects can be used, care can be taken to design a deliverysystem that targets such compounds to the site of affected tissue inorder to minimize potential damage to uninfected cells and, thereby,reduce side effects.

The data obtained from the cell culture assays and/or animal studies canbe used in formulating a range of dosage for use in humans. The dosageof such compounds can be within a range of circulating concentrationsthat includes the ED50 with little or no toxicity. The dosage can varywithin this range depending upon the dosage form employed and/or theroute of administration utilized. For certain exemplary compounds, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range that includes the IC50 (i.e., theconcentration of the test compound that achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma can be measured, for example, by high performance liquidchromatography.

An effective amount of the compositions, sufficient for achieving atherapeutic or prophylactic effect, can range from about 0.000001 mg perkilogram body weight per day to about 10,000 mg per kilogram body weightper day. The dosage ranges can be from about 0.0001 mg per kilogram bodyweight per day to about 100 mg per kilogram body weight per day. Asexamples, dosages can be about 1 mg/kg body weight or about 10 mg/kgbody weight every day, every two days, or every three days or within therange of about 1 to about 10 mg/kg every week, every two weeks, or everythree weeks. In certain exemplary embodiments, a single dosage ofpeptide can range from about 0.1 to about 10,000 micrograms per kg bodyweight. In certain exemplary embodiments, composition concentrations ina carrier range from about 0.2 to about 2000 micrograms per deliveredmilliliter can be administered. An exemplary treatment regime can entailadministration once per day or once a week. In certain therapeuticapplications, a relatively high dosage at relatively short intervals canbe required until progression of the disease is reduced and/orterminated, and/or until the subject shows partial or completeamelioration of symptoms of disease. Thereafter, the patient can beadministered a prophylactic regime.

In certain exemplary embodiments, a therapeutically effective amount ofa composition can be defined as a concentration of peptide at the targettissue of about 10⁻¹² to 10⁻⁶ about molar, e.g., approximately 10⁻⁷molar. This concentration can be delivered by systemic doses of about0.01 to about 100 mg/kg or equivalent dose by body surface area. Theschedule of doses can be optimized to maintain the therapeuticconcentration at the target tissue, such as by single daily or weeklyadministration, but also including continuous administration (e.g.,parenteral infusion and/or transdermal application).

In certain exemplary embodiments, the dosage of the composition can beprovided at a “low,” “mid,” or “high” dose level. In certain exemplaryembodiments, the low dose can be provided from about 0.01 to about 0.5mg/kg/h, such as from about 0.0001 to about 0.1 mg/kg/h. In certainexemplary embodiment, the mid-dose can provided from about 0.001 toabout 1.0 mg/kg/h, such as from about 0.01 to about 0.5 mg/kg/h. Incertain exemplary embodiments, the high dose can be provided from about0.005 to about 10 mg/kg/h, such as from about 0.01 to about 2 mg/kg/h.

In certain exemplary embodiments, certain factors can influence thedosage and/or timing required to effectively treat a subject, includingbut not limited to, the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and/or otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of the therapeutic compositionsdescribed herein can include a single treatment or a series oftreatments.

The mammal treated in accordance with certain exemplary methods can beany mammal, including, for example, farm animals, such as sheep, pigs,cows, and horses; pet animals, such as dogs and cats; and/or laboratoryanimals, such as rats, mice, and rabbits. In certain exemplaryembodiments, the mammal can be a human.

Certain exemplary embodiments provide for a biologically activecomposition of matter comprising a first D-biotin conjugated to a lysinelocated at the C-terminus of a water-soluble, cell-permeable,mitochondria-targeted peptide sequence, wherein:

-   -   the mitochondria-targeted peptide sequence comprises a minimum        of four amino acids and a maximum of six amino acids;    -   the mitochondria-targeted peptide sequence has a general        alternating aromatic-cationic motif;    -   the mitochondria-targeted peptide sequence is selected from:

D-Arg-L-(2′6′-dimethylTyr)-L-Lys-L-Phe-NH₂;D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys-NH₂; D-Trp-D-Arg-D-Trp-D-Lys-OH;(SEQ ID NO: 2) L-Trp-L-Arg-L-Trp-L-Lys-NH₂; D-Trp-D-Arg-D-Trp-D-Lys-NH₂;and/or (SEQ ID NO: 1) L-Trp-L-Arg-L-Trp-L-Lys-OH;

-   -   a second D-biotin is conjugated to an N-terminus α-amine of the        mitochondria-targeted peptide sequence;    -   the composition comprises one or more of:

D-Biotin-D-Arg-L-(2′6′-dimethylTyr)-L-Lys-L- Phe-NH₂; andD-Biotin-D-Trp-D-Arg-D-Trp-D-Lys-OH;

-   -   the first D-biotin is conjugated to an ε-amine of the lysine at        a C-terminus of the mitochondria-targeted peptide sequence;    -   the composition comprises one or more of:

D-Trp-D-Arg-D-Trp-D-Lys(biotinyl)-OH;L-Trp-L-Arg-L-Trp-L-Lys(biotinyl)-NH₂; andD-Arg-L-Tyr-D-Arg-L-Phe-L-Lys(biotinyl)-NH₂;

-   -   the first D-biotin is conjugated to an ε-amine of the lysine at        the C-terminus of the mitochondria-targeted peptide sequence and        a second D-biotin is conjugated to an N-terminus α-amine; and/or    -   the composition comprises one or more of:

D-Biotin-L-Trp-L-Arg-L-Trp-L-Lys(biotinyl)-NH₂; andD-Biotin-D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys(biotinyl)- NH₂.

Certain exemplary embodiments provide for a biologically activecomposition of matter comprising a first D-biotin conjugated to a lysinelocated at the N-terminus of a water-soluble, cell-permeable,mitochondria-targeted peptide sequence, wherein themitochondria-targeted peptide sequence:

-   -   comprises a minimum of four amino acids and a maximum of six        amino acids; and    -   has a general alternating aromatic-cationic motif.

Certain exemplary embodiments provide for a composition of matter,comprising:

-   -   a therapeutically effective formulation comprising:        -   one or more biologically active, water-soluble,            cell-permeable, mitochondria-targeted compounds selected            from a biotinylated polypeptide group;        -   a pharmaceutically acceptable carrier for each of the one or            more biologically active, water-soluble, cell-permeable,            mitochondria-targeted compounds;        -   one or more vitamins selected from:            -   vitamin B1 (thiamine);            -   vitamin B2 (riboflavin);            -   vitamin B3 (niacin, niacinamide);            -   vitamin B5 (pantothenic acid);            -   vitamin B6 (pyridoxine);            -   vitamin B7 (biotin);            -   vitamin B9 (folate);            -   vitamin B12 (cyanocobalamine); and            -   vitamin C (ascorbic acid);        -   one or more metabolic supplements selected from:            -   pyruvate;            -   carnitine;            -   acetylcarnitine;            -   creatine;            -   a-ketoglutarate;            -   a-lipoic acid;            -   coenzyme Q;            -   nicotinamide riboside; and            -   nicotinamide mononucleotide; and/or        -   one or more amino acids selected from:            -   leucine;            -   isoleucine;            -   valine;            -   glutamine;            -   serine;            -   arginine;            -   methionine;            -   tryptophan;            -   glycine;            -   trimethylglycine;            -   b-hydroxy-b-methylbutyrate; and            -   Taurine;    -   wherein:        -   each biologically active, water-soluble, cell-permeable,            mitochondria-targeted compound in the biotinylated            polypeptide group is defined by:            -   a plurality of amino acids arranged with a general                alternating aromatic-cationic motif;            -   a minimum of four amino acids and a maximum of six amino                acids;            -   a first D-biotin conjugated to a lysine located at the                C-terminus or the N-terminus of that biotinylated                polypeptide;        -   the biotinylated polypeptide group consists of:

D-Biotin-D-Arg-L-(2′6′-dimethylTyr)-L-Lys-L- Phe-NH₂;D-Biotin-D-Trp-D-Arg-D-Trp-D-Lys-OH;D-Trp-D-Arg-D-Trp-D-Lys(biotinyl)-OH;L-Trp-L-Arg-L-Trp-L-Lys(biotinyl)-NH₂;D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys(biotinyl)-NH₂;D-Biotin-L-Trp-L-Arg-L-Trp-L-Lys(biotinyl)-NH₂; andD-Biotin-D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys(biotinyl)- NH₂.

TABLE 1 identifies certain exemplary short, water-soluble, alternatingaromatic-cationic peptide sequences.

TABLE 1 SS-31 D-Arg L- L-Lys L-Phe NH₂ 2′6′dimethylTyr SPN02 D-Arg L-TyrD-Arg L-Phe L-Lys NH₂ SPN07 D-Trp D-Arg D-Trp D-Lys OH SPN10 L-Trp L-ArgL-Trp L-Lys NH₂ (SEQ ID NO: 1) SPN13 D-Trp D-Arg D-Trp D-Lys NH₂ SPN14L-Trp L-Arg L-Trp L-Lys OH (SEQ ID NO: 2)

The alternating aromatic-cationic peptide sequences useful in certainexemplary methods described herein can be chemically synthesized by anyof the methods described in the following US patent documents, each ofwhich is incorporated by reference herein in its entirety and/or for itsportion that describes or is relevant to synthesizing peptides and/orusing synthesized peptides:

-   -   U.S. Pat. No. 4,749,742;    -   U.S. Pat. Nos. 5,026,773;    -   U.S. Pat. No. 7,576,061;    -   U.S. Pat. No. 9,388,212;    -   U.S. Pat. No. 9,695,214;    -   U.S. patent Ser. No. 10/125,163;    -   US Patent Application Publication 2019/0202861;    -   US Patent Application Publication 2019/0015521; and    -   US Patent Application Publication 2012/0149868.

Certain exemplary alternating aromatic-cationic peptide sequences listedin Table 1 are water-soluble.

Certain exemplary alternating aromatic-cationic peptide sequences listedin Table 1 are water-soluble but can penetrate cell membranes.

Certain exemplary alternating aromatic-cationic peptide sequences listedin Table 1 are water-soluble but can penetrate mitochondrial outermembranes.

Biotin (FIG. 1A) can be conjugated to the alternating aromatic-cationicpeptide sequences at the N-terminus α-amine (FIG. 1B) or the ε-aminogroup of lysine at the C-terminus (FIG. 1C).

For certain exemplary embodiments, via chemically synthesis, biotin canbe conjugated to the polypeptide using one or more methods described inany of the following U.S. patent publications, each of which isincorporated by reference herein in its entirety and for its teachingsof such methods:

-   -   U.S. Pat. No. 5,391,711;    -   U.S. Pat. No. 5,416,016; and    -   U.S. Patent Application Publication: 2006/0149035.

Certain exemplary embodiments can preferentially label the α-aminoN-terminus in peptides with biotin. In certain exemplary embodiments,biotinylation can be readily accomplished by activating the carboxylgroup of biotin such that it reacts with free amino groups of thepeptide. Certain exemplary embodiments can a biotinylating reagent suchas D-biotin-N-hydroxy-succinimide ester or biotinyl-p-nitrophenyl estercan be used. The activated ester can react under mild conditions withamino groups to incorporate a biotin residue into the desired molecule.Certain exemplary embodiments can biotinylate macromolecules usingD-biotin-N-hydroxy-succinimide ester and/or can biotinylate an exogenousmolecule using biotinyl-ε-nitrophenyl ester as a biotinylating reagent.Other reagents such as D-biotinyl-ε-aminocaproic acidN-hydroxy-succinimide ester in which ε-aminocaproic acid serves as aspacer link to reduce steric hindrance can also be used by certainexemplary embodiments.

For certain exemplary embodiments, biotinylation can be performed at theε-amino group lysine to form biocytin. In certain exemplary embodiments,biocytin-containing peptides can be achieved via solid phase synthesisusing Fmoc-Lys(ε-biotinyl) at the C-terminal position, which can resultin Lys(biotinyl) or biocytin after undergoing acid cleavage of theresin. For example, to create biocytin, certain exemplary embodimentscan utilize one or more methods described in U.S. Pat. No. 2,720,527,which is incorporated by reference herein in its entirety And for itsteachings of such methods.

Certain exemplary peptide-conjugated biotin molecules and thecorresponding mitochondria-targeting peptide sequences are listed inTABLE 2.

TABLE 2 SS-31 D-Arg L-2′6′dimethylTyr L-Lys L-Phe NH₂ SPN05 biotin D-ArgL-2′6′dimethylTyr L-Lys L-Phe NH₂ SPN07 D-Trp D-Arg D-Trp D-Lys OH SPN08biotin D-Trp D-Arg D-Trp D-Lys OH SPN09 D-Trp D-Arg D-Trp D-Lys(bio) OHSPN10 L-Trp L-Arg L-Trp L-Lys NH₂ (SEQ ID NO: 1) SPN11 L-Trp L-Arg L-TrpL-Lys(bio) NH₂ SPN12 biotin L-Trp L-Arg L-Trp L-Lys(bio) NH₂ SPN02 D-ArgL-Tyr D-Arg L-Phe L-Lys NH₂ SPN15 D-Arg L-Tyr D-Arg L-Phe L-Lys(bio) NH₂SPN16 biotin D-Arg L-Tyr D-Arg L-Phe L-Lys(bio) NH₂

Cellular uptake and localization of peptide-conjugated biotin moleculeswere determined in HK-2 human renal epithelial cells and ARPE-19 humanretinal pigment epithelial cells (ATCC, Manassas, Va.). HK-2 cells werecultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 1 g/Lglucose and 10% fetal bovine serum (FBS), 100 units/ml penicillin and100 ug/ml streptomycin. ARPE-19 cells were cultured in DMEM/F12 mediumcontaining 1 g/L glucose and 10% fetal bovine serum (FBS), 100 units/mlpenicillin and 100 ug/ml streptomycin. Cells were incubated inhumidified incubator with 5% CO2 at 37° C. HK-2 cells and ARPE-19 cellswere seeded in 35 mm dish at an initial density of 5×10⁴ cells. FBS wasremoved from the culture medium for 3 days to deplete endogenous biotinin the cells. Cells were then incubated in serum-free media containingbiotin or peptide-conjugated biotin molecules for 12-hours (HK-2 cells)or 1-hour (ARPE-19 cells). All compounds were used at 1 uM.

Biotin uptake was determined using Streptavidin binding. Streptavidinhas high affinity for biotin. By using Alexa Fluor 594-conjugatedstreptavidin, it is possible to visualize biotin uptake usingfluorescence microscopy. Cells were fixed with 4% PFA for 10 min at RT,permeabilized in 0.1% triton X-100/PBS for 10 min at RT and incubatedwith 3.2 ug/ml Streptavidin-Alexa Fluor 594 (Jackson ImmunoResearch,West Grove, Pa.) and 5 ug/ml Hoechst 33342 (Novus Biologicals,Centennial, Colo.) for 30 min at RT. Hoechst 33342 is a fluorescentstain for labeling DNA and it is used for nuclear staining. Live cellimage buffer was added and Alexa-594 fluorescence (Ex/Em=591/614 nm) andHoechst fluorescence (Ex/Em=490/461 nm) was observed using the NikonEclipse Ti2 fluorescence microscope (100×oil objective). Ten randomfields from each sample were quantified by NIS-Elements Imaging Software(Nikon) for streptavidin fluorescence and normalized to Hoechstfluorescence to account for number of cells per field.

FIG. 2 shows representative microscopic images of HK-2 human renalepithelial cells after 12-hour incubation with 1 uM free biotin orselected peptide-conjugated biotin molecules from TABLE 2. Intracellularbiotin is visualized with streptavidin-AlexaFluor594 (red fluorescence).Nuclei are visualized with Hoechst 33342 dye (blue fluorescence) (1000×magnification). Minimal intracellular streptavidin fluorescence wasobserved when HK-2 cells were incubated with 1 uM biotin alone. Incontrast, all peptide-conjugated biotin compounds (SPN05, SPN08, SPN09,SPN11 and SPN12) showed intense red fluorescence indicating significantcellular uptake of biotin.

FIG. 3 is a graph quantifying biotin uptake in HK-2 human renalepithelial cells after 12-hour incubation with 1 uM biotin or selectedpeptide-conjugated biotin molecules from TABLE 2. Peptide-conjugatedbiotin molecules delivered significantly higher intracellular biotincontent compared to free biotin in HK-2 cells. Total streptavidinfluorescence normalized to cell number (Hoechst fluorescence) wascalculated from 10 random fields and averaged for each treatment. Alldata were then normalized to treatment with free biotin, with freebiotin arbitrarily set as 1.0. All peptide-conjugated biotin moleculesresulted in significantly higher intracellular biotin (*P<0.05,***P<0.001) compared to free biotin. The uptake of peptide-conjugatedbiotin molecules was 2 to 2.5 times higher than free biotin.

FIG. 4 shows representative microscopic images of ARPE-19 human retinalpigment epithelial cells after 1-hour incubation with 1 uM biotin orselected peptide-conjugated biotin molecules from TABLE 2. Intracellularbiotin is visualized with streptavidin-AlexaFluor594 (red fluorescence).Nuclei are visualized with Hoechst 33342 dye (blue fluorescence) (1000×magnification). All peptide-conjugated biotin compounds (SPN08, SPN09,SPN11, SPN12, SPN15 and SPN16) showed intense red staining with aperinuclear distribution.

FIG. 5 is a graph showing biotin uptake in ARPE-19 human retinal pigmentepithelial cells after 1-hour incubation with 1 uM biotin or selectedpeptide-conjugated biotin molecules from TABLE 2. Peptide-conjugatedbiotin molecules delivered significantly higher intracellular biotincontent compared to free biotin in ARPE-19 cells. The ratio ofstreptavidin fluorescence to Hoechst fluorescence was calculated from 10random fields and averaged for each treatment. All data were thennormalized to treatment with free biotin, with free biotin arbitrarilyset as 1.0. All peptide-conjugated biotin molecules resulted insignificantly higher intracellular biotin staining compared to freebiotin (***P<0.001). The uptake of peptide-conjugated biotin moleculeswas 1.5 to 2 times higher than free biotin.

These peptides enter cells by simple diffusion, and once in the cell,they are seen only in the mitochondria. As confirmed by FIG. 2 and FIG.4 for the identified SPN peptides. The distribution pattern shown forthose peptides (filamentous network starting around the nucleus) is verydistinct for mitochondria. Notice the nucleus is not stained, and thestaining is not everywhere inside the cell. This greatly reduces thechance of side effects by the compound acting on other cellularorganelles.

These results show that conjugation of biotin to the exemplary short,water-soluble, aromatic-cationic peptide sequences can significantlyincrease cell uptake of biotin. The microscopic images in FIG. 2 andFIG. 4 show that conjugation of biotin to SS-31, SPN02, SPN07 and SPN10(SEQ ID NO:1) can greatly enhance biotin uptake into two different celllines (kidney and retinal epithelial cells). These four short peptidesall follow an alternating aromatic-cationic motif, with SS-31 and SPN02having a “cationic-aromatic-cationic-aromatic” sequence order, whileSPN07 and SPN10 (SEQ ID NO:1) having an “aromatic-cationic-aromaticcationic” sequence order. All exemplary peptides are water-soluble.SPN02 demonstrates that a pentapeptide can work as well as tetrapeptidesand is water-soluble. SS-31 shows that non-naturally occurring aminoacids (2′6′-dimethylTyr) can be substituted for natural-occurring aminoacids. These peptides also support the use of Tyr, Phe or Trp as thearomatic amino acid, and the use of Arg or Lys as the cationic aminoacid. The exemplary examples demonstrate that the amino acids can be inD- or L-configuration, and amidation of the C-terminus has no impact onmitochondria-delivery but can improve peptide stability againstcarboxypeptidase degradation in vivo.

A generic peptide of 4-6 amino acids with an alternatingaromatic-cationic motif made of naturally-occurring or non-naturallyoccurring amino acids, in either D- or L-configuration, with or withoutC-terminus amidation, can serve as a water-soluble delivery vector toenhance cellular uptake of biotin in mammalian cells.

Example 2—Short Aromatic-Cationic Peptide Sequences Selectively DeliverBiotin to the Inner Mitochondrial Membrane

Certain exemplary alternating aromatic-cationic peptide sequences listedin Table 1 can selectively target and localize to the innermitochondrial membrane.

The perinuclear distribution pattern for all the peptide-conjugatedbiotin molecules (FIG. 2 and FIG. 4 ) suggests they are localized to thefilamentous mitochondrial network.

To demonstrate mitochondrial localization of peptide-conjugated biotinmolecules, ARPE-19 human retinal pigment epithelial cells were incubatedwith 1 uM SPN05, SPN12 or SPN15 for 2-hours in serum-free DMEM/F12media. Cells were fixed with 4% PFA at RT for 10 min and thenpermeabilized with 0.1% Triton X-100 at RT for 10 min. Cells wereblocked with 2% BSA (bovine serum albumin) at RT for 30 min, and thenincubated with primary antibody for cytochrome c oxidase subunit 4(COX4), a major protein complex expressed on the inner mitochondrialmembrane. The rabbit polyclonal COX4 antibody (Invitrogen PAS-29992,Waltham, Mass.) was used at 1:500 dilution in 2% BSA at 4° C. overnight.Cells were then incubated with secondary antibody (goat anti-rabbitAlexa Fluor 488, 1:500 dilution, Invitrogen A-11008) andStreptavidin-Alexa Fluor 594 (Jackson ImmunoResearch, West Grove, Pa.)and 5 ug/ml Hoechst 33342 (Novus Biologicals, Centennial, Colo.) for 30min at RT. Live cell image buffer was added and Alexa Fluor-594fluorescence), Alexa Fluor-488 and Hoechst fluorescence were observedusing the Nikon Eclipse Ti2 fluorescence microscope (100×oil objective).

FIG. 6 shows co-localization of peptide-conjugated biotin molecules withcytochrome c oxidase. Top panel are representative microscopic images ofSPN15 (staining red), cytochrome c oxidase (COX) (staining green), andthe merged image (yellow/orange). Bottom panel shows images of SPN05 andSPN12 merged with COX staining (yellow/orange). All images shown in 600×magnification.

These results demonstrate that short aromatic-cationic peptide sequencescan serve as delivery vectors to target biotin to mitochondria. COX4 isa subunit of cytochrome c oxidase that is the terminal complex (complexIV) of the electron transport chain. The COX complex is a majorregulation site for oxidative phosphorylation on the inner mitochondrialmembrane. The co-localization of biotin staining from SPN05, SPN12 andSPN15 with COX4 staining demonstrate that these short aromatic-cationicpeptide sequences can serve as mitochondria-targeting sequences todeliver biotin to the inner mitochondrial membrane.

Example 3—Peptide-Conjugated Biotin Molecules can Promote Cell GrowthUnder Prolonged Serum Starvation

Serum removal can cause decrease in cellular ATP, cell cycle arrest,and/or apoptosis. Serum deprivation can inhibit the ability of culturedcells to proliferate. Incubation with certain exemplarypeptide-conjugated biotin molecules can promote cell viability underserum-free conditions.

In the first model, HK-2 cells were cultured in 96-well plates inserum-free DMEM alone or in serum-free DMEM containing 10 nM of SPN11 orSPN12 for 11 days. Culture medium was replaced every 3 days.

Cell viability was measured using the Alamar Blue Cell ProliferationAssay (Bio-Rad), according to manufacturer's protocol. Alamar Blue usesresazurin to measure reducing power of living cells. The weaklyfluorescent resazurin is reduced to the highly fluorescent resorufinwhen reduced by cellular metabolism. Briefly, alamar blue reagent (10ul) was added to the culture medium and incubated for 1 h at 37° C.Fluorescence from resorufin (Ex/Em 530/590 nm) was detected using amicroplate reader (SpectraMax iD3, Molecular Devices).

FIG. 7 is a graph showing cell viability in HK-2 cells after 11 days ofserum starvation. Treatment with just 10 nM of SPN11 or SPN12significantly improved cell viability by 35% in the absence of serum(**P<0.005).

In the second model, ARPE-19 cells were grown in serum-free DMEM in theabsence or presence of peptide-conjugated biotin molecules for 30 days.Serum-free media was supplemented with 10 nM of SPN12, SPN15 or SPN16compounds (all at 10 nM) was replaced every 3 days. Culture medium wasreplaced every 3 days. FIG. 8 are representative microscopic images showthat all three SPN compounds increased cell number after prolonged serumdeprivation.

FIG. 9 shows that treatment with SPN12, SPN15 and SPN16 significantlydoubled cell viability after 30 days of serum deprivation compared tocontrol. Data represent mean±SEM from 6 samples for each treatment(**P<005; ***P<0.001, compared to control).

These results show that addition of just 10 nM of peptide-conjugatedbiotin molecules can significantly double cell survival in serum-freeconditions. These results demonstrate that exemplarymitochondria-targeted biotin molecules are biologically active (i.e.,they alter cell biology) to protect cell survival in at least twodifferent mammalian cell systems.

Example 4—Peptide-Conjugated Biotin Molecules Increased Cellular ATPContent in Serum-Free and Nutrient-Deprived Cultures

Serum removal can cause decrease in cellular ATP. Certain exemplarypeptide-conjugated biotin molecules can increase cellular ATP levels inserum-deprivation and/or nutrient-deprivation conditions.

Human renal epithelial cells (HK-2) were cultured in DMEM containing 1g/L glucose and 10% fetal bovine serum (FBS), 100 units/ml penicillin,and 100 ug/ml streptomycin. Cells were incubated in humidified incubatorwith 5% CO2 at 37° C. HK-2 cells were seeded in 96-well culture platesat an initial density of 5×10³ cells. FBS was removed from the culturemedium and cells were incubated in serum-free DMEM alone (control group)or containing 10 nM of biotin or peptide-conjugated biotin molecules for7 days. All treatments were carried out with N=6 in each experiment. Theculture medium was changed every 3 days.

Human retinal pigment epithelial cells (ARPE-19) were cultured inDMEM/F12 media containing 1 g/L glucose and 10% fetal bovine serum(FBS), 100 units/ml penicillin, and 100 ug/ml streptomycin. Cells wereincubated in humidified incubator with 5% CO2 at 37° C. ARPE-19 cellswere seeded in 96-well culture plates at an initial density of 5×10³cells. FBS was removed from the culture medium and cells were incubatedin serum-free DMEM/F12 alone (control group) or containing 10 nM biotinor different peptide-conjugated biotin molecules for 7 days. Alltreatments were carried out with N=5-6 in each experiment. The culturemedium was changed every 3 days.

ATP was measured using the ApoSENSOR ATP Bioluminescence Assay Kit(BioVision) according to manufacturer's protocol. This kit utilizesluciferase to catalyze the formation of light from ATP and luciferin.Briefly, cells were treated with 100 ul of Nuclear Releasing Buffer for5 min at RT with gentle shaking. 10 ul of ATP Monitoring Enzyme wasadded to cell lysate, and luminescence was measured using a microplatereader (SpectraMax iD3, Molecular Device). ATP levels were normalized tothe serum-free DMEM control group (arbitrarily set to 1.0).

All peptide-conjugated biotin molecules resulted in higher ATPconcentrations compared to free biotin in HK-2 cells after 7 days inserum-free conditions (FIG. 10 ). Data shown are mean±SEM from 6 samplesper treatment. SPN08, SPN09, SPN11, SPN12, SPN15, and SPN16 elevated ATPlevels in serum-free conditions by 35%-100%, compared to free biotin(**P<0.005; ***P<0.001).

The peptide-conjugated biotin molecules also significantly increased ATPconcentration in ARPE-19 cells after 7 days of serum starvation. Datashown are mean±SEM from 6 samples per treatment. The peptide-conjugatedbiotin molecules elevated ATP levels by 60-75%, compared to free biotin(***P<0.001) (FIG. 11 ).

The second model uses an extreme starvation model whereby HK-2 cellswere cultured in 96-well plates in serum-free 5% DMEM in PBS(phosphate-buffered saline) for 3 days. Cells were treated with 10 nMfree biotin or peptide-conjugated biotin molecules. All treatments werecarried out with N=6 in each experiment.

All peptide-conjugated biotin molecules resulted in significantly higherATP concentrations compared to free biotin under extreme starvation.Data shown are mean±SEM from 6 samples per treatment. (*P<0.05;**P<0.01; ***P<0.001), with SPN11, and SPN12 doubling ATP content (FIG.12 ).

These results demonstrate that exemplary mitochondria-targeted biotinmolecules are biologically active promoting mitochondrial ATP synthesisunder serum- and nutrient-deprived conditions in at least two differentmammalian cell systems.

Example 5—Peptide-Conjugated Biotin Molecules can Restore MitochondrialPotential and Prevent Mitochondrial Fragmentation Caused by SerumStarvation

Serum deprivation can induce mitochondrial depolarization which precedesthe reduction in ATP synthesis. Certain exemplary peptide-conjugatedbiotin molecules can restore mitochondrial potential in cells culturedin serum-free medium.

Electron transfer along the electron transport chain on the innermitochondrial membrane results in the pumping of protons from themitochondrial matrix to the inter-membrane space. This generates anelectrical potential across the inner mitochondrial membrane, and theproton gradient serves to drive the ATP synthase (complex V) to produceATP from ADP. Withdrawal of serum or nutrients leads to decline inmitochondrial potential and reduced ATP production.

Human retinal pigment epithelial cells (ARPE-19) were cultured inDMEM/F12 media containing 1 g/L glucose and 10% FBS, 100 units/mlpenicillin, and 100 ug/ml streptomycin. Cells were incubated inhumidified incubator with 5% CO2 at 37° C. ARPE-19 cells were seeded in35 mm glass plates at an initial density of 5×10⁴ cells. FBS was removedfrom the culture medium for 3 days to deplete endogenous biotin. Cellswere then incubated in serum-free DMEM/F12 alone (control group) orDMEM/F12 containing 1 uM of SPN12 or SPN15 for 2 hours.

To determine mitochondrial potential, live cells were incubated with 5nM tetramethylrhodamine methyl ester (TMRM, #70017, Biotium, Fremont,Calif.) in DMEM without phenol red. TMRM is a potential-dependentfluorescent dye (Ex/Em=548/573). TMRM accumulates in negatively chargedpolarized mitochondria and can be detected as red fluorescence. Totalmitochondria were imaged with a mitochondrial potential-independentfluorescent dye (100 nM MitoView Green; Ex/Em=490/523; #70054, Biotium,Fremont Calif.). Hoechst 33342 (10 ug/ml, Novus Biologicals, Centennial,Colo.) was added to stain nuclei. Live cells were covered with phosphatebuffer and imaged using the Nikon Eclipse Ti fluorescent microscopyusing the 60×water objective.

FIG. 13 (top panel) are representative fluorescent microscopic images ofARPE-19 cells cultured for 3 days in DMEM/F12 with 10% FBS. FIG. 13(bottom panel) are representative images of ARPE-19 cells cultured for 3days in serum-free DMEM/F12. TMRM (red) shows dramatic loss ofmitochondrial potential in serum-free condition when compared to serumcontrol. The mitogreen (green) stain also shows that serum-freecondition caused mitochondria to fragment and aggregate in a perinuclearpattern with no clear filamentous network. When the images are merged,mitochondrial depolarization can be seen all cells without serum.

FIG. 14 are representative fluorescent microscopic images of ARPE-19cells cultured for 3 days in 10% FBS (serum control) or in serum-freemedium (serum-free) alone or after 2 hours incubation with 1 uM of SPN12or SPN15. The images shown are merged images of TMRM (red) and MitoGreen(green). Two-hour incubation with SPN compounds was sufficient torestore mitochondrial potential in all cells and recover the filamentousmitochondrial network in ARPE-19 cells after 3 days of serum starvation.

These results show that mitochondria are completely depolarized after 3days of serum starvation and the mitochondria network in the cells arefragmented. The peptide-conjugated biotin molecules (SPN12 and SPN15)can rescue mitochondrial potential in a matter of 2 hours in serumstarvation. These results confirm that biotin conjugated to thearomatic-cationic peptide sequences is targeted to the innermitochondrial membrane and are biologically active in protecting theelectrical potential across the inner mitochondrial membrane.

Example 6—Peptide-Conjugated Biotin Molecules are More Effective thanFree Biotin in Wound Repair

Certain exemplary peptide-conjugated biotin molecules can acceleratewound healing in cell cultures. The in vitro scratch assay is an easyand well-developed method to measure cell proliferation and migrationover a “wound area” in vitro. This assay involves creating a “scratch”in a cell monolayer with a pipette tip and then examining the rate atwhich cells proliferate and migrate to close the scratch.

HK-2 cells were cultured in DMEM (1 g/L glucose) with 10% FBS, 100units/ml penicillin, and 100 ug/ml streptomycin. 3×10⁵ cells were platedper well in 6-well plates in DMEM at 37° C. in a humidified incubatorwith 5% CO2 for one day prior to the experiment. To mimic themicroenvironment of wounds in vitro, serum-free medium was used in thescratch assay. On the day of the scratch assay, the medium was replacedwith serum-free DMEM, and a line scraped across the cell monolayer usinga p1000 pipette tip. Cells were washed to remove cell debris andreplaced with DMEM alone (control), or DMEM containing 10 nM biotin orpeptide-conjugated biotin molecules.

The scratch area was examined immediately (day 0) using a Nikon EclipseTi2 fluorescence microscope. Six different field were captured for eachsample using 4×objective. The scratch area (cell-free zone) wascalculated using ImageJ software, an open-source Java image processingprogram inspired by NIH Image (National Institute of Health). Thescratch area was re-examined after 24 hours (day 1), and the area wasnormalized to the area determined on day 0 for the same sample. Allresults were calculated as change from no-treatment control and averagedfor the six fields.

FIG. 15 are representative microscopic images showing treatment with 10nM SPN11, or SPN12 greatly accelerated the closure of the scratch inHK-2 cells 24 hours after mechanical scratch. The scratch areasdetermined after 24 hours are normalized to the area determinedimmediately after application of the scratch (Day 0) for each treatmentgroup. Biotin had a small effect on reducing scratch area whereas thescratch area was almost completely closed by SPN11 and SPN12 in 24hours.

FIG. 16 summarizes the effect of biotin, SPN11 and SPN12 on reducingscratch area 24 hours after application of mechanical scratch in HK-2cells. All treatments significantly accelerated the closure of thescratch area (***P<0.001, compared to control). The biotin-conjugatedpeptides (SPN11 and SPN12) are twice as effective compared to biotin inreducing scratch area (P<0.001).

FIG. 17 shows that the other peptide-conjugated biotin molecules, SPN15and SPN16 also significantly accelerated the closure of the scratch areaafter 24 hours in another experiment in HK-2 cells (***P<0.001) and arethree times more effective compared to free biotin (P<0.001).

The peptide-conjugated biotin molecules (SPN12, SPN15 and SPN16) alsoaccelerated wound healing in ARPE-19 cells (FIG. 18 ). FIG. 19 is agraph summarizing the effects of the peptide-conjugated biotin moleculeson scratch area (**P<0.01; ***P<0.001). The peptide-conjugated biotinmolecules were significantly more effective (1.5 to 2-fold) compared tofree biotin (P<0.001).

These results demonstrate that not only are the peptide-conjugatedbiotin molecules biologically-active, they are superior to free biotinin promoting wound healing in vitro.

Example 7—Peptide-Conjugated Biotin Molecules can Promote CellProliferation in Wound Area

Re-epithelialization of a wound area require proliferation of cells atthe wound edge, which can be impaired in most wounds due to lack ofblood flow to deliver nutrients and/or growth factor(s) that can benecessary for cell proliferation. Cell proliferation in the wound edgecan be monitored by proliferating cell nuclear antigen (PCNA) staining.To mimic the microenvironment of wounds in vitro, serum-free medium wasused for the scratch assay in cell cultures.

Proliferating cells were identified by immunostaining with antibody toproliferating cell nuclear antigen (PCNA) according to standardprocedures as presented in IHC World. PCNA staining was performed usingthe same plate of cells after determination of scratch area. HK-2 cellswere fixed with ethanol:methanol (1:1) for 30 min at −20° C.,permeabilized with 0.1% Triton X-100/PBS for 10 min at RT, blocked with2% goat serum for 30 min at RT, and incubated with primary mouseanti-PCNA antibody (Dako Agilent, Santa Clara, Calif.) and secondarygoat anti-mouse IgG-BI for 30 min at RT. Cells were then incubated withStreptavidin-Alexa Fluor 594 (Jackson ImmunoResearch, West Grove, Pa.)and Hoechst (Novus Biologicals, Centennial, Colo.) and imaged with aNikon Eclipse Ti2 Fluorescent microscope (20× objective). All cellnuclei stain blue with Hoechst, but nuclei of proliferating cells stainred. The number of proliferating cells per field was quantified by theintensity of red nuclear stain and normalized to intensity of bluenuclear stain (determined by Nikon NIS-Elements Imaging Software).

FIG. 20 shows representative microscopic images (×40 magnification) ofHK-2 human renal epithelial cell monolayers 24 hours after mechanicalscratch in serum-free medium only (control), or in serum-free mediumcontaining exemplary SPN compounds (10 nM). Cells positive forproliferating cell nuclear antigen are shown in red. All other nucleiare shown in blue. Compared to free biotin, SPN11 and SPN12 increasedthe number of proliferating cells at the edge of the scratch.

FIG. 21 is a graph summarizing the number of proliferating cells in HK-2human renal epithelial cell monolayers 24 hours after mechanical scratchin serum-free medium only (control), or in serum-free mediumsupplemented with 10 nM biotin or peptide-conjugated biotin molecules.The number of proliferating cells were normalized to all nuclei. Theeffect of biotin was small and did not reach statistical significance.Incubation with SPN11, and SPN12 doubled the number of proliferatingcells at the edge of the scratch compared to control or biotin(***P<0.001).

FIG. 22 shows representative microscopic images (40× magnification) ofARPE-19 human retinal pigment epithelial cell monolayers 24 hours aftermechanical scratch in serum-free medium only (control), or in serum-freemedium supplemented with 10 nM free biotin or peptide-conjugated biotinmolecules. Cells positive for proliferating cell nuclear antigen areshown in red. All other nuclei are shown in blue. Incubation with theSPN08, SPN09, SPN11, or SPN12 increased the number of proliferatingcells at the edge of the scratch, while 10 nM biotin had no effect oncell proliferation.

FIG. 23 is a graph summarizing the number of proliferating cells inARPE-19 human retinal pigment epithelial cell monolayers 24 hours aftermechanical scratch in serum-free medium only (control), or in serum-freemedium containing 10 nM biotin or peptide-conjugated biotin molecules.The number of proliferating cells were normalized to all nuclei.Incubation with each of SPN09, SPN11, SPN12, SPN15, and SPN16significantly increased the percent of proliferating cells at the edgeof the scratch by 35-100%, but biotin itself was without effect(*P<0.05; **P<0.01; ***P<0.001, compared to control).

These results show that the peptide-conjugated biotin molecules are moreeffective than biotin alone in promoting cell proliferation in a woundsetting, and this is likely due to increased delivery of biotin tomitochondria.

Example 8—Peptide-Conjugated Biotin Molecules Protect MitochondrialPotential of Cells at the Wound Edge

Rapidly dividing cells can need ATP to support the synthesis ofimportant building blocks. Tissue injury can cause rapid mitochondrialdepolarization that compromises ATP production and/or results in celldeath. The loss of ATP can further impair cell proliferation and/ortissue repair. Mitochondrial potential can be monitored in the cells atthe edge of the scratch area using the cell-permeablepotential-dependent dye TMRM (tetramethylrhodamine methyl ester). TMRMcan accumulate in negatively charged polarized mitochondria and can bedetected as red fluorescence.

To mimic the microenvironment of wounds in vitro, serum-free medium wasused in the scratch assay. On the day of the scratch assay, the mediumwas replaced with serum-free DMEM, and a line scraped across the HK-2cell monolayer using a p1000 pipette tip. Cells were washed to removedebris and replaced with DMEM alone (control), or DMEM supplemented with10 nM free biotin or peptide-conjugated biotin molecules for 24 hours.HK-2 cells were then incubated with 5 nM TMRM in DMEM without phenol redand incubated for 30 min. Cells were then covered with live cell imagebuffer and imaged using Nikon Eclipse Ti2 fluorescent microscope (20×objective).

FIG. 24 shows representative microscopic images (200× magnification) ofmitochondrial potential in HK-2 cells 24 hours after mechanical scratchin serum-free medium only (control), or in serum-free medium containing10 nM biotin, SPN11, or SPN12. Mitochondrial potential is detected withTMRM, which stains red, while nuclei stain blue with Hoechst. Undercontrol conditions, few cells at the edge of the scratch showed TMRMstaining, indicating that many cells underwent mitochondrialdepolarization Addition of 10 nM SPN11, or SPN12 increased the number ofcells with good mitochondrial potential, whereas biotin had no effect.

FIG. 25 is a graph summarizing the ratio of mitochondrial potential (redfluorescence) normalized to nuclear number (blue fluorescence) in HK-2cells 24 hours after mechanical scratch in serum-free medium only(control), or in serum-free medium containing 10 nM biotin orpeptide-conjugated biotin molecules. Incubation with SPN11, or SPN12significantly increased mitochondrial potential in cells at the edge ofthe scratch (***P<0.001, compared to control). The effect of free biotindid not reach statistical significance.

These results demonstrate that the biotin-conjugated peptides (SPN11 andSPN12) are superior to free biotin in preserving mitochondrial potentialin a wound setting because of their targeted delivery to the innermitochondrial membrane. This can translate to better increase in ATPproduction and cell proliferation.

Example 9—Uptake and Distribution of SPN15 to Mouse Retina afterIntraperitoneal Administration

To demonstrate that the peptide-conjugated biotin molecules can beadministered in vivo, we have determined the uptake and distribution ofSPN15 in a mouse following intraperitoneal (ip) administration.

SPN15 (30 mg/kg) was administered ip to a 13-month-old male mouse(C57BL/6J strain). At 2-hours after administration, the mouse wasanesthetized with an overdose of ketamine/xylazine, and the eyesenucleated. After the cornea and lens were removed, the eyecup was fixedfor 1.25 hours in 4% paraformaldehyde in 0.1M Tris buffer. The eyecupwas then rinsed 3 times for 10 minutes each in Tris buffer and thencryoprotected in 10%, 20%, and 30% sucrose before sectioning at 30 umwith a Leica 3050 cryostat. Sections were stored at −20° C. until used.

To label biotin on SPN15, sections were incubated with Streptavidinconjugated AlexaFluor 594 (1:500) from Jackson ImmunoResearch, WestGrove, Pa.). ToPro 3 (nuclear stain, 1:1000, Molecular Probes) was usedto stain cell nuclei. Mitochondria localization was determined using anantibody to Cox IV (cytochrome c oxidase subunit 4; PA529992, 1:300,Invitrogen) and Mitotracker Red (1:1500 in Tris, Invitrogen). Allincubations were carried out in microwave (150 W) except for Cox IV andMitotracker labeling, which was done with primary antibody overnight at4° C. followed by secondary antibody (AF488 conjugated donkeyanti-rabbit, Jackson Immunoresearch). Sections were imaged using theOlympus Fluoview 300 with helium, argon and neon lasers using 40×oilimmersion lens at a resolution of 1024×1024.

FIG. 26 (left panel) is a cryostat section from a control mouse withoutpeptide administration showing nuclei labeled with TOPRO (blue) in thedifferent layers of the retina, where OS=outer segment; IS=innersegment; ONL=outer nuclear layer; OPL=outer plexiform layer; INL=innernuclear layer; IPL=inner plexiform layer; RGC=retinal ganglion cell.

FIG. 26 (right panel) is a cryostat section from a control mouse stainedfor COX4, which is a protein expressed on the inner mitochondrialmembrane (green), Mitotracker Red, which is a fluorescent that labelsmitochondria (red), and TOPRO, which labels nuclei (blue). COX4 stainingfor mitochondria is clearly seen in the photoreceptor IS with theirabundant mitochondria, and the INL, IPL and the RGC. The staining ofMitotracker Red in the photoreceptor OS is surprising because there areno mitochondria in the OS.

FIG. 26 (middle panel) is a cryostat section from a mouse 2 hours afterSPN15 administration. SPN15 is clearly taken up in the different layersof the retina as visualized by Streptavidin Alexa Fluor 594 (red)staining. The SPN15 staining is concentrated in the inner and outerplexiform layers (IPL and OPL), where synapses between ganglion, bipolarand amacrine cells in the inner nuclear layer (INL) and photoreceptors.Streptavidin staining co-localizes with COX4 staining in the RGC, IPL,INL, and the photoreceptor IS. An exception is the intense streptavidinstaining in the photoreceptor OS where there is absence of COX4staining. It is unclear why SPN15 is so heavily distributed to the OSwhich contains densely-packed disks responsible for light transduction.

FIG. 27 is an enlargement of the photoreceptor IS and OS. The retinalpigment epithelium (RPE) is a single layer of cells above the OS. SPN15staining can clearly be seen in the RPE cells.

These results show that SPN15 is rapidly absorbed after ipadministration in a living mouse and is widely distributed throughoutthe retina. There are two sources of blood supply to the mammalianretina: the central retinal artery and the choroidal blood vessels. Thecentral retinal artery starts at the optic nerve to supply the innerretinal layers. The choroid blood flow is vital for the maintenance ofthe outer retina (photoreceptors and retinal pigment epithelium (RPE).The streptavidin staining indicates that SPN15 is distributed throughboth vascular systems.

SPN15 staining is concentrated in the inner and outer plexiform layers(IPL and OPL). The outer nuclear layer (ONL) contains cell bodies of therods and cones, and the inner nuclear layer (INL) contains cell bodiesof ganglion cells, horizontal cells and amacrine cells. The OPL is wheresynapses between rods and cones, and vertically running bipolar cellsand horizontally oriented cells occur. The IPL functions as a relaystation for the bipolar cells to the ganglion cells, and this is wherethe message concerning the visual image is transmitted to the brainalong the optic nerve.

SPN15 is also concentrated in the inner segment (IS) that containsaggregates of very long thin mitochondria to support metabolism of bothrod and cone photoreceptors. The high concentration of SPN15 in the ISis expected due to the density of mitochondria. These mitochondria playa role in the biosynthesis of the numerous lipid disks in the outersegment (OS) that contains the visual pigment molecules (rhodopsin) forvisual transduction. The very high concentration of SPN15 in the OS isunexpected as there is a complete absence of mitochondria. This isconfirmed by the lack of staining for COX4 in the OS. The staining of OSby Mitotracker has been reported but the reason is unclear and wouldrequire further investigation. The concentration of SPN15 in the OS canbe important for maintenance of disk stability.

The OS disks suffer light-induced oxidative damage and are normallyphagocytized by the retinal pigment epithelium (RPE) and degraded bylysosomal degradation, allowing rhodopsin to be recycled, and thegeneration of ketone bodies which can be used as metabolic fuel byphotoreceptors. The RPE is a single layer of cell between the OS and thechoroid that also forms the blood-retinal barrier. The distribution ofSPN15 in the RPE layer is highlighted in FIG. 27 .

These findings confirm that SPN15 can be distributed to areas of highmitochondrial density in the retina after systemic administration. Theysuggest that SPN15 and other peptide-conjugated biotin molecules can bebeneficial for numerous ophthalmic diseases. The targeting of RPEsuggests SPN15 can improve mitochondrial bioenergetics in aging andcombat age-related macular degeneration and diabetic retinopathy. Thisis supported by the results that SPN compounds preserve mitochondrialpotential, increase ATP synthesis, improve cell viability, and increasecell proliferation in ARPE-19 cells, a human retinal pigment epithelialcell line. SPN15 also can increase viability of retinal ganglion cellsunder increased pressure from glaucoma and result in optic nerve damage.

Definitions

When the following phrases are used substantively herein, theaccompanying definitions apply. These phrases and definitions arepresented without prejudice, and, consistent with the application, theright to redefine these phrases via amendment during the prosecution ofthis application or any application claiming priority hereto isreserved. For the purpose of interpreting a claim of any patent thatclaims priority hereto, each definition in that patent functions as aclear and unambiguous disavowal of the subject matter outside of thatdefinition.

-   -   a—at least one.    -   activity—an action, act, step, and/or process or portion thereof    -   adapt—to design, make, set up, arrange, shape, configure, and/or        make suitable and/or fit for a specific purpose, function, use,        and/or situation.    -   add—to join and/or unite (something) to something else in order        to increase the size, quantity, effect, and/or scope.    -   administer—to give and/or apply.    -   alcohol—any of a class of chemical compounds having the general        formula ROH, where R represents an alkyl group and —OH a        hydroxyl group, as in methyl alcohol, CH3OH, or ethyl alcohol,        C2H5OH.    -   alternating—designating or relating to every other one of a        series.    -   amine—any of a group of organic compounds of nitrogen, such as        ethylamine,    -   C2H5NH2, that may be considered ammonia derivatives in which one        or more hydrogen atoms have been replaced by a hydrocarbon        group.    -   amino acid—a compound in which at least one amino group and at        least one carboxyl group are bound to the same carbon skeleton        and the nitrogen atom of the amino group may form part of a        ring, such compounds including the L- and D-isomers of the        natural amino acids.    -   and—in conjunction with.    -   and/or—either in conjunction with or in alternative to.    -   antibiotics—a substance, such as penicillin or erythromycin,        produced by and/or derived from certain microorganisms,        including fungi and bacteria, that can destroy or inhibit the        growth of other microorganisms, especially bacteria. Antibiotics        are widely used in the prevention and treatment of infectious        diseases.    -   anticonvulsant—any of a class of drugs used to prevent or        abolish convulsions.    -   antioxidant—substances that reduce the production of reactive        oxygen species, inhibit the oxidation of other substances,        substances that retard the deterioration of other substances by        oxidation, and/or scavengers of free radical species, reactive        oxygen species, hydroxyl radical species, oxidized lipids,        and/or lipid peroxidation products.    -   any—one, some, every, and/or all without specification.    -   apparatus—an appliance or device for a particular purpose.    -   approximately—about and/or nearly the same as, including for        each value in a series of two or more numerical values; within        an acceptable error for a particular value as determined by a        person having ordinary skill in the art, which depends in part        on how the value is measured or determined; within one standard        deviation; when no particular margin of error (e.g., a standard        deviation to a mean value given in a chart or table of data) is        recited, the range that encompasses the recited value and would        be included by rounding up or down to the recited value as well,        taking into account significant figures; and/or within ±20%,        15%, 10%, or 5% of the specified value.    -   aromatic—an organic compound having an unsaturated ring        containing alternating double and single bonds, including those        compounds having a benzene ring.    -   arrange—to dispose in a particular order.    -   associate—to join, connect together, and/or relate.    -   at—in, on, and/or near.    -   at least—not less than, and possibly more than, which applies to        each value in any series of values that the phrase “at least”        precedes.    -   be—to exist in actuality.    -   biologically-active—configured to alter cell biology.    -   biotin—a crystalline, water-soluble vitamin, C10H16O3N2S, of the        vitamin B complex, sometimes referred to as vitamin B7 and/or        vitamin H.    -   biotinidase—an enzyme, which in humans is encoded by the BTD        gene, that readily cleaves and/or breaks down biotin amides,        releasing free biotin and the amine, and which the main        substrate of which is biocytin, or biotin linked to lysine;        biotinidase is also capable of breaking apart biotin esters.    -   biotinylated—a polypeptide having a biotin molecule located at        one or more of its terminals.    -   C-terminus—(also known as the carboxyl-terminus,        carboxy-terminus, C-terminal tail, C-terminal end, or        COOH-terminus) the end of an amino acid chain (protein or        polypeptide), terminated by a free carboxyl group (—COOH).    -   can—is capable of, in at least some embodiments.    -   carrier—a substance to which an active ingredient and/or agent        is added as a way of applying and/or transferring that active        ingredient and/or agent.    -   cationic—an ion or group of ions having a positive charge and        characteristically moving toward the negative electrode in        electrolysis.    -   cause—to bring about, provoke, precipitate, produce, elicit, be        the reason for, result in, and/or effect.    -   caused by—resulting from.    -   cell—the smallest structural unit of an organism that is capable        of independent functioning, consisting of cytoplasm, usually one        nucleus, and various other organelles, all surrounded by a        semipermeable cell membrane.    -   cell-permeable—capable of freely moving passively into and out        of cells.    -   cellular—of, relating to, or consisting of cells.    -   chemically-defined medium—those mediums in which all components        in the medium are identified with exact concentrations and the        medium contains no added animal or human serum, growth factors,        hormones, etc.    -   composition of matter—a combination, reaction product, compound,        mixture, formulation, material, and/or composite formed by a        human and/or automation from two or more substances and/or        elements.    -   compound—a pure, macroscopically homogeneous substance        consisting of atoms or ions of two or more different elements in        definite proportions that cannot be separated by physical means.        A compound usually has properties unlike those of its        constituent elements.    -   comprising—including but not limited to.    -   conceive—to imagine, conceptualize, form, and/or develop in the        mind.    -   configure—to design, arrange, set up, shape, and/or make        suitable and/or fit for a specific purpose, function, use,        and/or situation.    -   conjugate—to link two chemical compounds together via a covalent        bond.    -   conservative substitution—the substitution of an amino acid in a        polypeptide with a functionally, structurally, and/or chemically        similar natural or unnatural amino acid, such as the following        groups each contain natural amino acids that are conservative        substitutions for one another:        -   1) Glycine (Gly/G), Alanine (Ala/A);        -   2) Isoleucine (Ile/I), Leucine (Leu/L), Methionine (Met/M),            Valine (Val/V);        -   3) Phenylalanine (Phe/F), Tyrosine (Tyr/Y), Tryptophan            (Trp/W);        -   4) Serine (Ser/S), Threonine (Thr/T), Cysteine (Cys/C);        -   5) Asparagine (Asn/N), Glutamine (Gln/Q);        -   6) Aspartic acid (Asp/D), Glutamic acid (Glu/E); and/or        -   7) Arginine (Arg/R), Lysine (Lys/K), Histidine (His/H).    -    and/or the following groups, which each contain natural amino        acids that are conservative substitutions for one another:        -   1) non-polar: Ala, Val, Leu, Ile, Met, Pro (proline/P), Phe,            Trp;        -   2) hydrophobic: Val, Leu, Ile, Phe, Trp;        -   3) aliphatic: Ala, Val, Leu, Ile;        -   4) aromatic: Phe, Tyr, Trp, His;        -   5) uncharged polar or hydrophilic: Gly, Ala, Ser, Thr, Cys,            Asn, Gln, Tyr;        -   6) aliphatic hydroxyl- or sulfhydryl-containing: Ser, Thr,            Cys;        -   7) amide-containing: Asn, Gln;        -   8) acidic: Asp, Glu;        -   9) basic: Lys, Arg, His; and/or        -   10) small: Gly, Ala, Ser, Cys.    -    and/or the following groupings:        -   1) hydrophobic: Val, Leu, Ile, Met, Phe, Trp;        -   2) aromatic: Phe, Tyr, Trp, His;        -   3) neutral hydrophilic: Gly, Ala, Ser, Thr, Cys, Asn, Gln;        -   4) acidic: Asp, Glu;        -   5) basic: Lys, Arg, His; and/or        -   6) residues that influence backbone orientation: Pro, Gly.    -   consisting—relating to a closed group (and its legal        equivalents) that otherwise excludes anything not listed.    -   consume—to eat and/or drink; to take into the body by the mouth        for digestion and/or absorption.    -   containing—including but not limited to.    -   convert—to transform, adapt, and/or change.    -   corresponding—related, associated, accompanying, similar in        purpose and/or position, conforming in every respect, and/or        equivalent and/or agreeing in amount, quantity, magnitude,        quality, and/or degree.    -   create—to bring into being.    -   culture media—a nutritive substance, such as an agar gel or        liquid medium, in which cultures of bacteria, fungi, animal        cells, or plant cells are grown and/or cultivated for scientific        purposes.    -   deficiency—a state and/or condition that deviates from a desired        state and/or condition.    -   define—to establish the meaning, relationship, outline, form,        and/or structure of;    -   and/or to precisely and/or distinctly describe and/or specify.    -   delivery—an act of conveying and/or transferring.    -   derive—to receive, obtain, and/or produce from a source and/or        origin.    -   determine—to find out, obtain, calculate, decide, deduce,        ascertain, and/or come to a decision, typically by        investigation, reasoning, and/or calculation.    -   device—a machine, manufacture, and/or collection thereof    -   each—every one of a group considered individually.    -   effective—sufficient to bring about, provoke, elicit, and/or        cause.    -   embodiment—an implementation, manifestation, and/or concrete        representation.    -   estimate—(n) a calculated value approximating an actual        value; (v) to calculate and/or determine approximately and/or        tentatively.    -   excessive—exceeding a normal, usual, reasonable, and/or proper        limit.    -   exemplary—serving as an example, instance, and/or illustration,        but not necessarily preferred or advantageous over other        embodiments or features.    -   first—an initial cited element of a set.    -   formulation—a medicinal preparation administered in a specific        form, such as a tablet, linctus, ointment, or injection.    -   from—used to indicate a source, origin, and/or location thereof    -   general alternating—having the identified sequence or its        inverse, e.g., aromatic-cationic or cationic-aromatic, such as a        peptide sequence with A-C-A-C-A-C or C-A-C-A-C-A residues (where        A stands for aromatic and C for cationic).    -   generate—to create, produce, give rise to, and/or bring into        existence.    -   greater than—at least.    -   group—(n.) a number of individuals and/or things considered        together because of one or more similarities; (v.) to associate        a number of individuals or things such that they are considered        together and/or caused to have similar properties.    -   having—including but not limited to.    -   health—the overall condition of an organism at a given time;        soundness, especially of body and/or mind; freedom from disease,        injury, disorder, and/or abnormality.    -   improve—to change to a better state and/or condition.    -   inadequate—insufficient.    -   including—having, but not limited to, what follows.    -   initialize—to prepare something for use and/or some future        event.    -   injury—damage and/or harm done to and/or suffered by a person        and/or thing.    -   install—to connect or set in position and prepare for use.    -   intake—the act and/or instance of taking in, ingesting, and/or        receiving.    -   intestinal malabsorption—defective and/or inadequate absorption        of nutrients from the intestinal tract.    -   intramuscular—within a muscle.    -   intranasal—within the nose.    -   intravenous—within a vein.    -   is—to exist in actuality.    -   lysine—a crystalline, basic, essential amino acid,        H2N(CH2)4CH(NH2)COOH, produced chiefly from many proteins by        hydrolysis.    -   maintenance—an activity relating to restoring and/or preserving        performance of an item and/or system.    -   mammal—any of various warm-blooded vertebrate animals of the        class    -   Mammalia, including humans, characterized by a covering of hair        on the skin and, in the female, milk-producing mammary glands        for nourishing the young.    -   mammalian—of and/or relating to a mammal.    -   maximum—having a greatest value.    -   may—is allowed and/or permitted to, in at least some        embodiments.    -   medical condition—diseases, injuries, disorders, and/or        abnormalities of the body and/or mind.    -   medication—a substance adapted to relieve at least one symptom        of and/or cure a medical condition.    -   metabolic—of and/or relating to metabolism.    -   metabolic supplement—naturally occurring compounds that enhance        energy production.    -   metabolism—the chemical processes occurring within a living cell        or organism that are necessary for the maintenance of life. In        metabolism some substances are broken down to yield energy for        vital processes while other substances, necessary for life, are        synthesized.    -   method—one or more acts that are performed upon subject matter        to be transformed to a different state or thing and/or are tied        to a particular apparatus, said one or more acts not a        fundamental principal and not pre-empting all uses of a        fundamental principal.    -   minimum—having a lowest value.    -   mitigate—to make less severe, serious, or painful.    -   mitochondria—a spherical or elongated organelle in the cytoplasm        of nearly all eukaryotic cells, containing genetic material and        many enzymes important for cell metabolism, including those        responsible for the conversion of food to usable energy.    -   mitochondria-targeted—indicates that an indicated substance        (e.g., biotin) is transported to mitochondria such that, once        inside the cell, the substance will be preferentially localized        to mitochondria, and not substantially distributed to other        cellular organelles or membranes.    -   more—a quantifier meaning greater in size, amount, extent,        and/or degree.    -   motif—a recurrent pattern either of molecular sequence, usually        of nucleotides or amino acids in proteins, or of molecular        structure that usually corresponds to specific biological        activity.    -   N-terminus—(also known as the amino-terminus, NH2-terminus,        N-terminal end or amine-terminus) is the start of a protein or        polypeptide referring to the free amine group (—NH2) located at        the end of a polypeptide. Within a peptide, the amine group is        bonded to another carboxylic group in a protein to make it a        chain, but since the end amino acid of a protein is only        connected at the carboxy-end, the remaining free amine group is        called the N-terminus.    -   no—an absence of and/or lacking any.    -   one—being and/or amounting to a single unit, individual, and/or        entire thing, item, and/or object.    -   operable—practicable and/or fit, ready, and/or adapted to be put        into its intended use and/or service.    -   or—a conjunction used to indicate alternatives, typically        appearing only before the last item in a group of alternative        items.    -   oral—of and/or relating to the mouth.    -   organ—a differentiated part of an organism, such as an eye,        wing, or leaf, that performs a specific function.    -   patient—a mammalian subject, such as a human subject.    -   peptide—any of various natural or synthetic compounds (including        amino acid salts such as pharmaceutically acceptable salts)        containing two or more amino acids covalently joined by at least        one peptide and/or amide bond that links the carboxyl group of        one amino acid to the amino group of another.    -   per—for each and/or by means of    -   pharmaceutically acceptable—a substance (e.g., an active        ingredient or an excipient) that is suitable for use in contact        with the tissues and organs of a subject without excessive        irritation, allergic response, immunogenicity, and/or toxicity,        is commensurate with a reasonable benefit/risk ratio, is        effective for its intended use, and/or is compatible with the        other ingredients of any composition that comprises the        substance; that which is useful in preparing a pharmaceutical        composition and is generally safe, non-toxic, and neither        biologically nor otherwise undesirable and includes that which        is acceptable for veterinary use as well as human pharmaceutical        use; and/or that which is approved and/or approvable by a        regulatory agency of the Federal and/or a state government        and/or the corresponding agency in countries other than the        United States, and/or that is listed in the U.S. Pharmacopoeia        and/or other generally recognized pharmacopoeia for use in        animals, and more particularly, in humans.    -   plurality—the state of being plural and/or more than one.    -   polypeptide—a chain of amino acids linked together by peptide        bonds and having a molecular weight of up to about 10,000.    -   polypeptides—generally peptides and proteins, for which herein        the left-hand end of the polypeptide sequence is referred to as        the “amino (N)-terminus”, and the right-hand end of the sequence        is referred to as the “carboxyl I-terminus”.    -   portion—a part, component, section, percentage, ratio, and/or        quantity that is less than a larger whole.    -   pre-—a prefix that precedes an activity that has occurred        beforehand and/or in advance.    -   predetermine—to determine, decide, and/or establish in advance.    -   preserve—to store safely for later use.    -   prevent—to impede, avert, resist, hinder, stop, and/or keep from        happening.    -   probability—a quantitative representation of a likelihood of an        occurrence.    -   product—something produced by human and/or mechanical effort.    -   project—to calculate, estimate, or predict.    -   promote—to contribute to the progress and/or growth of; to        further, advance, promote, and/or market.    -   protein—a linked sequence of amino acid residues containing 50        or more amino acid residues.    -   provide—to furnish, supply, give, and/or make available.    -   range—a measure of an extent of a set of values and/or an amount        and/or extent of variation.    -   ratio—a relationship between two quantities expressed as a        quotient of one divided by the other.    -   receive—to get as a signal, take, acquire, and/or obtain.    -   recommend—to suggest, praise, commend, and/or endorse.    -   reduce—to make and/or become lesser and/or smaller.    -   regeneration—regrowth of lost and/or destroyed parts and/or        organs.    -   remove—to eliminate, remove, and/or delete, and/or to move from        a place or position occupied.    -   repair—to restore to a desired condition.    -   repeat—to do again and/or perform again.    -   repeatedly—again and again; repetitively.    -   request—to express a desire for and/or ask for.    -   result—(n.) an outcome and/or consequence of a particular        action, operation, and/or course; (v.) to cause an outcome        and/or consequence of a particular action, operation, and/or        course.    -   said—when used in a system or device claim, an article        indicating a subsequent claim term that has been previously        introduced.    -   second—a cited element of a set that follows an initial element.    -   select—to make a choice or selection from alternatives.    -   selected—chosen from a plurality of alternatives.    -   sequence—an ordered set.    -   serum-free—lacking the clear yellowish fluid obtained upon        separating whole blood into its solid and liquid components        after it has been allowed to clot.    -   set—a related plurality.    -   solution—a homogeneous mixture of two or more substances, which        may be solids, liquids, gases, or a combination of these.    -   species—a class of individuals and/or objects grouped by virtue        of their common attributes and assigned a common name; a        division subordinate to a genus.    -   storage—the act of storing or the state of being stored    -   store—to set aside, reserve, deposit, secure, and/or put away        for future use.    -   subcutaneous—slightly below the skin.    -   subject—an animal, including but not limited to a mammal, such        as a primate (e.g., a human, a chimpanzee, or a monkey), a        rodent (e.g., a rat, a mouse, a guinea pig, a gerbil, or a        hamster), a lagomorph (e.g., a rabbit), a bovine (e.g., a        cattle), a suid (e.g., a pig), a caprine (e.g., a sheep), an        equine (e.g., a horse), a canine (e.g., a dog), and/or a feline        (e.g., a cat).    -   sublingual—situated beneath and/or on the underside of the        tongue.    -   substantially—to a great extent and/or degree.    -   suffer—to feel pain or distress; to sustain injury and/or harm;        to endure, be afflicted with, and/or be ill with; to be        accurately diagnosed with.    -   supplement—(n.) a product containing one or more vitamins,        herbs, enzymes, amino acids, and/or other ingredients, that is        taken to supplement one's diet, as by providing a missing        nutrient.    -   system—a collection of mechanisms, devices, machines, articles        of manufacture, processes, compositions of matter, data, and/or        instructions, the collection designed to perform one or more        specific functions.    -   target—to interact with.    -   therapeutically—of or relating to the medical treatment of a        disease, injury, disorder, and/or abnormality.    -   therapeutically effective amount—an amount of a substance that,        when administered to a subject, is sufficient to prevent, reduce        the risk of developing, delay the onset of, slow the progression        of, and/or cause regression of the medical condition being        treated, and/or to alleviate to some extent the medical        condition and/or one or more symptoms and/or complications of        that condition, at least in some fraction of the subjects taking        that substance, and/or to elicit the biological and/or medical        response of a cell, tissue, organ, system, animal, and/or human        that is sought by a researcher, veterinarian, medical doctor,        and/or clinician.    -   tissue—an aggregation of morphologically similar cells and        associated intercellular matter acting together to perform one        or more specific functions in the body.    -   tissue regeneration—re-growth of tissue through cell        proliferation that completely restores portion of damaged tissue        to their normal state.    -   tissue repair—the restoration of tissue architecture and        function after an injury, said restoration encompassing tissue        regeneration and tissue replacement.    -   tissue replacement—those types of healing where the damaged        tissues are repaired by laying down connective tissue or scar        tissue.    -   topical—a localized area of the body, and typically the skin.    -   transdermal—through or by way of the skin.    -   transform—to change in measurable: form, appearance, nature,        and/or character.    -   transmit—to send as a signal, provide, furnish, and/or supply.    -   transport—to convey and/or move from one place to another.    -   treat—to alleviate, ameliorate, inhibit the progress of,        reverse, prevent, and/or abrogate a medical condition and/or one        or more causes, symptoms, and/or complications associated with        the condition; to handle and/or deal with someone and/or        something.    -   uptake—to intake, consume, and/or use.    -   use—to put into service.    -   used—employed in accomplishing something.    -   via—by way of and/or utilizing.    -   vitamin—any of various fat-soluble or water-soluble organic        substances that are essential in minute amounts for normal        growth and activity of living organisms, such substances        synthesized by bacteria and/or plants and/or obtained by animals        chiefly in their diet; and/or an organic substance, other than a        protein, carbohydrate, or fat, that is an essential constituent        of the food of an animal to which the vitamin is administered,        e.g., B group vitamins including B1 (thiamine), B2 (riboflavin),        B3 (niacin, niacinaide), B5 (pantothenic acid), B6 (pyridoxine),        B7 (biotin), B9 (folate), and B12 (cyanocobalamine), Vitamin C        (ascorbic acid), vitamin D and vitamin K.    -   water-soluble—capable of being dissolved in water.    -   weight—a force with which a body is attracted to Earth or        another celestial body, equal to the product of the object's        mass and the acceleration of gravity; and/or a factor and/or        value assigned to a number in a computation, such as in        determining an average, to make the number's effect on the        computation reflect its importance, significance, preference,        impact, etc.    -   wherein—in regard to which; and; and/or in addition to.    -   with—accompanied by.    -   zone—a region and/or volume having at least one predetermined        boundary.

Note

Various substantially and specifically practical and useful exemplaryembodiments of the claimed subject matter are described herein,textually and/or graphically, including the best mode, if any, known tothe inventor(s), for implementing the claimed subject matter by personshaving ordinary skill in the art. References herein to “in oneembodiment”, “in an embodiment”, or the like do not necessarily refer tothe same embodiment.

Any of numerous possible variations (e.g., modifications, augmentations,embellishments, refinements, and/or enhancements, etc.), details (e.g.,species, aspects, nuances, and/or elaborations, etc.), and/orequivalents (e.g., substitutions, replacements, combinations, and/oralternatives, etc.) of one or more embodiments described herein mightbecome apparent upon reading this document to a person having ordinaryskill in the art, relying upon his/her expertise and/or knowledge of theentirety of the art and without exercising undue experimentation. Theinventor(s) expects any person having ordinary skill in the art, afterobtaining authorization from the inventor(s), to implement suchvariations, details, and/or equivalents as appropriate, and theinventor(s) therefore intends for the claimed subject matter to bepracticed other than as specifically described herein. Accordingly, aspermitted by law, the claimed subject matter includes and covers allvariations, details, and equivalents of that claimed subject matter.Moreover, as permitted by law, every combination of the herein describedcharacteristics, functions, activities, substances, and/or structuralelements, and all possible variations, details, and equivalents thereof,is encompassed by the claimed subject matter unless otherwise clearlyindicated herein, clearly and specifically disclaimed, or otherwiseclearly unsuitable, inoperable, or contradicted by context.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate one or moreembodiments and does not pose a limitation on the scope of any claimedsubject matter unless otherwise stated. No language herein should beconstrued as indicating any non-claimed subject matter as essential tothe practice of the claimed subject matter.

Thus, regardless of the content of any portion (e.g., title, field,background, summary, description, abstract, drawing figure, etc.) ofthis document, unless clearly specified to the contrary, such as viaexplicit definition, assertion, or argument, or clearly contradicted bycontext, with respect to any claim, whether of this document and/or anyclaim of any document claiming priority hereto, and whether originallypresented or otherwise:

-   -   there is no requirement for the inclusion of any particular        described characteristic, function, activity, substance, or        structural element, for any particular sequence of activities,        for any particular combination of substances, or for any        particular interrelationship of elements;    -   no described characteristic, function, activity, substance, or        structural element is “essential”; and    -   within, among, and between any described embodiments:        -   any two or more described substances can be mixed, combined,            reacted, separated, and/or segregated;        -   any described characteristic, function, activity, substance,            component, and/or structural element, or any combination            thereof, can be specifically included, duplicated, excluded,            combined, reordered, reconfigured, integrated, and/or            segregated;        -   any described interrelationship, sequence, and/or dependence            between any described characteristics, functions,            activities, substances, components, and/or structural            elements can be omitted, changed, varied, and/or reordered;        -   any described activity can be performed manually,            semi-automatically, and/or automatically;        -   any described activity can be repeated, performed by            multiple entities, and/or performed in multiple            jurisdictions.

The use of the terms “a”, “an”, “said”, “the”, and/or similar referentsin the context of describing various embodiments (especially in thecontext of the following claims) are to be construed to cover both thesingular and the plural, unless otherwise indicated herein or clearlycontradicted by context.

The terms “comprising,” “having,” “including,” and “containing” are tobe construed as open-ended terms (i.e., meaning “including, but notlimited to,”) unless otherwise noted.

When any number or range is described herein, unless clearly statedotherwise, that number or range is approximate. Recitation of ranges ofvalues herein are merely intended to serve as a shorthand method ofreferring individually to each separate value falling within the range,unless otherwise indicated herein, and each separate value and eachseparate sub-range defined by such separate values is incorporated intothe specification as if it were individually recited herein. Forexample, if a range of 1 to 10 is described, that range includes allvalues therebetween, such as for example, 1.1, 2.5, 3.335, 5, 6.179,8.9999, etc., and includes all sub-ranges therebetween, such as forexample, 1 to 3.65, 2.8 to 8.14, 1.93 to 9, etc., even if those specificvalues or specific sub-ranges are not explicitly stated.

When any phrase (i.e., one or more words) appearing in a claim isfollowed by a drawing element number, that drawing element number isexemplary and non-limiting on claim scope.

No claim or claim element of this document is intended to invoke 35 USC112(f) unless the precise phrase “means for” is followed by a gerund.

Any information in any material (e.g., a United States patent, UnitedStates patent application, book, article, web page, etc.) that has beenincorporated by reference herein, is incorporated by reference herein inits entirety to its fullest enabling extent permitted by law yet only tothe extent that no conflict exists between such information and theother definitions, statements, and/or drawings set forth herein. In theevent of such conflict, including a conflict that would render invalidany claim herein or seeking priority hereto, then any such conflictinginformation in such material is specifically not incorporated byreference herein. Any specific information in any portion of anymaterial that has been incorporated by reference herein that identifies,criticizes, or compares to any prior art is not incorporated byreference herein.

Applicant intends that each claim presented herein and at any pointduring the prosecution of this application, and in any application thatclaims priority hereto, defines a distinct patentable invention and thatthe scope of that invention must change commensurately if and as thescope of that claim changes during its prosecution. Thus, within thisdocument, and during prosecution of any patent application relatedhereto, any reference to any claimed subject matter is intended toreference the precise language of the then-pending claimed subjectmatter at that particular point in time only.

Accordingly, every portion (e.g., title, field, background, summary,description, abstract, drawing figure, etc.) of this document, otherthan the claims themselves and any provided definitions of the phrasesused therein, is to be regarded as illustrative in nature, and not asrestrictive. The scope of subject matter protected by any claim of anypatent that issues based on this document is defined and limited only bythe precise language of that claim (and all legal equivalents thereof)and any provided definition of any phrase used in that claim, asinformed by the context of this document when reasonably interpreted bya person having ordinary skill in the relevant art.

What is claimed is:
 1. A method for producing human cells, the methodcomprising: proliferating the human cells in a culture medium, wherein:the culture medium comprises an effective amount of: one or morebiologically active, water-soluble, cell-permeable,mitochondria-targeted compounds selected from a biotinylated polypeptidegroup, each biologically active, water-soluble, cell-permeable,mitochondria-targeted compound in the biotinylated polypeptide groupdefined by: a plurality of amino acids arranged with a generalalternating aromatic-cationic motif; a minimum of four amino acids and amaximum of six amino acids; a first D-biotin conjugated to a lysinelocated at the C-terminus or the N-terminus of that biotinylatedpolypeptide, wherein the biotinylated polypeptide is optionally amidatedat the C-terminus.
 2. The method according to claim 1, wherein theculture medium comprises: an effective amount of one or more vitaminsselected from a vitamin group consisting of: vitamin B1 (thiamine);vitamin B2 (riboflavin); vitamin B3 (niacin, niacinamide); vitamin B5(pantothenic acid); vitamin B6 (pyridoxine); and vitamin B7 (biotin). 3.The method according to claim 1, wherein the culture medium comprises:an effective amount of one or more amino acids selected from an aminoacid group consisting of: L-Leucine; L-Isoleucine; L-Valine;L-Glutamine; L-Serine; L-Arginine; L-Methionine; L-Tryptophan; andGlycine.
 4. The method according to claim 1, wherein the culture mediumcomprises: an effective amount of one or more metabolic supplementsselected from a metabolic supplement group consisting of: pyruvate;carnitine; acetylcarnitine; creatine; α-ketoglutarate; α-lipoic acid;nicotinamide riboside; and nicotinamide mononucleotide.
 5. The methodaccording to claim 1, wherein the biotinylated polypeptide groupconsists of: (SPN09) D-Trp-D-Arg-D-Trp-D-Lys(biotinyl)-OH; (SPN11)L-Trp-L-Arg-L-Trp-L-Lys(biotinyl)-NH₂; (SPN15)D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys(biotinyl)-NH₂; (SPN12)D-Biotin-L-Trp-L-Arg-L-Trp-L-Lys(biotinyl)-NH₂; and (SPN16)D-Biotin-D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys(biotinyl)- NH₂.


6. The method according to claim 1, wherein: the culture medium is freeof animal serum.
 7. The method according to claim 1, wherein: theculture medium is free of animal-derived products.
 8. The methodaccording to claim 1, wherein: the culture medium is free of humanserum.
 9. The method according to claim 1, wherein: the culture mediumis free of human-derived products.
 10. The method according to claim 1,wherein: the culture medium is free of growth factors.
 11. The methodaccording to claim 1, further comprising: increasing intracellularbiotin in the human cells by 50 percent via exposure of the human cellsto the one or more biologically active, water-soluble, cell-permeable,mitochondria-targeted biotinylated polypeptides in the culture medium(compared to exposure to the human cells to free biotin in a referenceculture medium).
 12. The method according to claim 1, furthercomprising: increasing biotin in mitochondria in the human cells by 50percent via exposure of the human cells to the one or more biologicallyactive, water-soluble, cell-permeable, mitochondria-targetedbiotinylated polypeptides in the culture medium (compared to exposure tothe human cells to free biotin in a reference culture medium).
 13. Themethod according to claim 1, further comprising: adding the one or morebiologically active, water-soluble, cell-permeable,mitochondria-targeted biotinylated polypeptides to the culture mediumduring ex vivo expansion of the human cells.
 14. The method according toclaim 1, further comprising: increasing viability of the human cells by35% (compared to a serum-free control culture medium lacking the one ormore biologically active, water-soluble, cell-permeable,mitochondria-targeted biotinylated polypeptides) when the culture mediumis serum-free for 11 days.
 15. The method according to claim 1, furthercomprising: increasing viability of the human cells by 100% (compared toa serum-free control culture medium lacking the one or more biologicallyactive, water-soluble, cell-permeable, mitochondria-targetedbiotinylated polypeptides) when the culture medium is serum-free for 30days.
 16. The method according to claim 1, further comprising:increasing ATP concentration in the human cells by 30% (compared to aserum-free control culture medium lacking the one or more biologicallyactive, water-soluble, cell-permeable, mitochondria-targetedbiotinylated polypeptides but containing comparable concentration offree biotin) when the culture medium is serum-free for 7 days.
 17. Themethod according to claim 1, further comprising: increasingproliferation and migration of the human cells to reduce a cell-freearea in a serum-free cell culture by 50% within 24 hours (compared to aserum-free control culture medium lacking the one or more biologicallyactive, water-soluble, cell-permeable, mitochondria-targeted compoundsselected from the biotinylated polypeptide group).
 18. The methodaccording to claim 1, further comprising: increasing proliferation andmigration of the human cells to reduce a cell-free area in a serum-freecell culture by 25% within 24 hours (compared to a serum-free controlculture medium containing free biotin but lacking the one or morebiologically active, water-soluble, cell-permeable,mitochondria-targeted compounds selected from the biotinylatedpolypeptide group).
 19. The method according to claim 1, furthercomprising: promoting proliferation of the human cells adjacent to acell-free area in a serum-free cell culture by 35% within 24 hours(compared to a serum-free control culture medium lacking the one or morebiologically active, water-soluble, cell-permeable,mitochondria-targeted compounds selected from the biotinylatedpolypeptide group).
 20. The method according to claim 1, furthercomprising: increasing average mitochondrial potential of the humancells adjacent to a cell-free area in a serum-free cell culture by 100%within 24 hours (compared to a serum-free control culture medium lackingthe one or more biologically active, water-soluble, cell-permeable,mitochondria-targeted compounds selected from the biotinylatedpolypeptide group).
 21. The method according to claim 1, furthercomprising: increasing average mitochondrial potential of the humancells adjacent to a cell-free area in a serum-free cell culture by 60%within 24 hours (compared to a serum-free culture medium containing freebiotin but lacking the one or more biologically active, water-soluble,cell-permeable, mitochondria-targeted compounds selected from thebiotinylated polypeptide group).
 22. A method for improving ex vivoviability of mammalian primary cells, the method comprising exposing theprimary cells to one or more compounds selected from a biotinylatedpolypeptide group consisting of: (SPN08)D-Biotin-D-Trp-D-Arg-D-Trp-D-Lys-OH; (SPN09)D-Trp-D-Arg-D-Trp-D-Lys(biotinyl)-OH; (SPN11)L-Trp-L-Arg-L-Trp-L-Lys(biotinyl)-NH₂; (SPN15)D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys(biotinyl)-NH₂; (SPN12)D-Biotin-L-Trp-L-Arg-L-Trp-L-Lys(biotinyl)-NH₂; and (SPN16)D-Biotin-D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys(biotinyl)- NH₂.


23. The method according to claim 22, wherein the primary cells comprisepancreatic islet cells.
 24. A method for improving proliferation ofmammalian cells, the method comprising adding to a serum-free culturemedium one or more compounds selected from a biotinylated polypeptidegroup consisting of: (SPN08) D-Biotin-D-Trp-D-Arg-D-Trp-D-Lys-OH;(SPN09) D-Trp-D-Arg-D-Trp-D-Lys(biotinyl)-OH; (SPN11)L-Trp-L-Arg-L-Trp-L-Lys(biotinyl)-NH₂; (SPN15)D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys(biotinyl)-NH₂; (SPN12)D-Biotin-L-Trp-L-Arg-L-Trp-L-Lys(biotinyl)-NH₂; and (SPN16)D-Biotin-D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys(biotinyl)- NH₂.


25. The method according to claim 24, wherein the mammalian cellscomprise primary cells.
 26. The method according to claim 24, whereinthe mammalian cells comprise T cells.
 27. The method according to claim24, wherein the mammalian cells comprise mesenchymal stem cells.
 28. Themethod according to claim 24, further comprising: culturing themammalian cells in the culture medium.
 29. A method for promotingsurvival of a mammalian organ and/or tissue, the method comprisingstoring the mammalian organ and/or tissue in a preservation solutioncomprising an effective amount of one or more compounds selected from abiotinylated polypeptide group consisting of: (SPN08)D-Biotin-D-Trp-D-Arg-D-Trp-D-Lys-OH; (SPN09)D-Trp-D-Arg-D-Trp-D-Lys(biotinyl)-OH; (SPN11)L-Trp-L-Arg-L-Trp-L-Lys(biotinyl)-NH₂; (SPN15)D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys(biotinyl)-NH₂; (SPN12)D-Biotin-L-Trp-L-Arg-L-Trp-L-Lys(biotinyl)-NH₂; and (SPN16)D-Biotin-D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys(biotinyl)- NH₂.


30. A method according to claim 29, wherein the mammalian organ and/ortissue comprises a kidney, liver, heart, lungs, pancreas, skin, bloodvessel, intestine, cornea, and/or trachea organ and/or tissue.
 31. Amethod for improving graft function and/or graft survival after organtransplantation, the method comprising administering to a transplantrecipient one or more compounds selected from a biotinylated polypeptidegroup consisting of: (SPN08) D-Biotin-D-Trp-D-Arg-D-Trp-D-Lys-OH;(SPN09) D-Trp-D-Arg-D-Trp-D-Lys(biotinyl)-OH; (SPN11)L-Trp-L-Arg-L-Trp-L-Lys(biotinyl)-NH₂; (SPN15)D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys(biotinyl)-NH₂; (SPN12)D-Biotin-L-Trp-L-Arg-L-Trp-L-Lys(biotinyl)-NH₂; and (SPN16)D-Biotin-D-Arg-L-Tyr-D-Arg-L-Phe-L-Lys(biotinyl)- NH₂.